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Dynein- and Microtubule-Mediated Translocation of Adenovirus Serotype 5 Occurs after Endosomal Lysis
Abstract
Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.
... Actin disruption with cytochalasin treatment was shown to impair internalization of the virus, since the actin network is required for endocytosis of Ad5 (Patterson and Russell 1983;Li et al. 1998b). However, once inside the cell, the integrity of the actin network is not required for the nuclear targeting of Ad5 (Leopold et al. 2000). One the other hand, the disruption of microtubules with nocodazole has no effect on virus entry or on endosomal escape (Suomalainen et al. 1999), but does impair nuclear targeting, leading to gene expression defect (Mabit et al. 2002;Suomalainen et al. 1999). ...
... Thus, intact microtubules are required for nuclear targeting of the Ad5. However, once docked at the NE, the disruption of microtubules has been shown to not impair the localization of Ad5, remaining associated to the nucleus (Leopold et al. 2000). After translocation to the nuclear envelope, the integrity of the microtubule network is not required anymore. ...
... The role of such interaction is not well established. Several studies point to targeting and accumulation of Ad5 capsids at the MTOC (or at the mitotic spindle) prior to nuclear translocation (Suomalainen et al. 1999;Leopold et al. 2000). Nevertheless, there is no direct evidence whether a passage through the MTOC is required for every capsid to be delivered to the NPC. ...
Adenoviruses (AdV) are DNA viruses that replicate in the nucleus of their host cell. Due to the limited coding capacity, they have to take advantage of cellular mechanisms in order to perform their infection cycle. During entry, AdV particles use the microtubule transport machinery to reach the nucleus. AdVs interact with the microtubule motor dynein to be transported towards the nuclear compartment, where they dock to Nuclear Pore Complexes (NPCs), structures embedded into the nuclear envelop (NE). Once at the NPC, viral capsids disassemble to finally release and import their genome. Microtubule unloading, nuclear translocation and genome import of AdVs involve components of the nucleocytoplasmic transport machinery. However, the exact mechanism used by the virus to reach the NPC remains unclear. Nucleocytoplasmic transport involves different components and is tightly regulated. The active transport of cargoes is mediated by import and export factors interacting with RanGTP. The major cellular export factor CRM1 is known to be essential for targeting of AdVs to the NE. Pharmacological inhibition of CRM1 with Leptomycin B leads to the accumulation of AdVs at the centrosome, the major Microtubule Organisation Centre (MTOC) in mammalian cells. We thus investigated the role of CRM1 leading to AdV genome delivery. We analysed the interaction of AdV with the MTOC and observed that the absence of cytoplasmic factors and disruption of microtubules did not impair their accumulation at the MTOC. We identified and characterized a mutant of CRM1, functional for physiological export but inducing a strong delay in AdV NE translocation. We used live cell-imaging to analyse infections in mitotic cells, revealing a role of CRM1 in genome release from the capsid. Moreover, we identified a potential viral partner of CRM1 among the AdV genome associated core proteins, the Terminal Protein. Terminal protein contains a nuclear export signal and is a CRM1 export substrate. Taken together, our data highlight a possible role of CRM1 as an essential mediator for the complete dismantling of AdV capsid, promoting genome release and genome import.
... Cell-specific nuclear translocation has been achieved by incorporating promoter sequences which are recognized by transcription factors unique to smooth muscle cells (Vacik at al. 1999). A recent study suggests that highly condensed PEI-DNA complexes (nanocomplexes) efficiently enter the nucleus by motor-protein dependent microtubule transport (Suh at al. 2003), a nuclear entry pathway which has also been exploited by several viruses (Leopold at al. 2000;Diefenbach at al. 2002). ...
... antibody; (c) nuclear entry by motor protein-dependent microtubule transport -which is used by several viruses (Leopold et al. 2000;Diefenbach et ai. 2002), and recently by PEI-DNA nanocomplexes (Suh et ai. ...
An upregulation of neurotrophic receptor (p75NTR) expression has been associated with a number of neurodegenerative disorders, cancers and atherosclerosis, and often directly contributes to disease progression. Hence p75-targeted gene delivery vectors should prove useful for the treatment of these diseases, either by expression of relevant therapeutic genes or by blocking expression of p75NTR in affected cells. Three phage display libraries were screened on cells over-expressing human p75NTR to isolate peptides with high affinity for this receptor. Two 7-mer peptides - VNLQNPY and VYARSMN - were identified as strong and highly specific binders to cells expressing p75NTR when displayed on bacteriophage M13. The p75-specificity of these peptides was further confirmed by phage immunostaining and confocal microscopy. Both peptides were subsequently synthesised with a DNA-targeting polylysine tail (K16) and incorporated into a liposome vector harbouring the luciferase reporter gene. Compared with un targeted control vectors, transfection efficiencies to p75-positive cells were between 5-10 fold higher, in particular with the VYARSMN peptide. Gene delivery to p75-negative cells was generally reduced by 50%. The identified peptide may thus be useful for designing synthetic and viral vectors for targeted gene transfer to degenerating neurons, vascular plaques or cancer cells in patients with AD, atherosclerosis and cancer respectively.
... After endosomal escape, viral particles engage with the MT network in order to reach the nucleus. The cellular motor protein dynein is responsible for transporting the capsid towards the minus end of MT (Suomalainen et al., 1999;Leopold et al., 2000). It was shown that dynein directly interacts with the viral hexon via the intermediate chains (IC1 and IC2) and light intermediate chain 1 (LIC1); mechanism that differs from that of physiological cargo (Bremner et al., 2009). ...
... Cellular motor proteins mediate transport of adenoviral particles towards the nucleus. They reach the microtubule organizing center (MTOC) in about 1 hour post infection (Suomalainen et al., 1999;Leopold et al., 2000). At this point, viral particles need to engage with nuclear factors to approach the nuclear membrane, as deduced from experiments performed in enucleated epithelial cells, showing an accumulation of incoming virions at the MTOC when the nucleus is removed (Bailey et al., 2003). ...
Nuclear delivery of viral genomes is an essential step for nuclear replicating DNA viruses such asAdenovirus (AdV). AdV particles reach the nuclear pore complex (NPC) in the form of genomecontaining, partially disassembled capsids, through a poorly understood CRM1-dependent mechanism.These capsids exceed the NPC size limit and therefore, they must disassemble at the NPC to releasethe viral genome. Nuclear import of DNA cargos is not a physiological process. Consequently, AdVneed to divert the cellular transport machinery for nuclear genome delivery. The NPC is a multiproteincomplex consisting of nucleoporins (Nups). The Nup358/RanBP2 is the major component ofthe cytoplasmic filaments of the NPC and serves as binding platform for factors includingkaryopherins (i.e Importin-β, CRM1) and the small GTPase Ran. Selective transport of cargo throughthe NPC is mediated by karyopherins, which recognize specific signals within the cargos and facilitatetheir transport in a RanGTP-dependent regulated manner. We identified that Nup358-depleted cellsreduce nuclear import efficiency of the AdV genome. Indeed, we observed that karyopherins are ratelimitingfor AdV genome import under these conditions and we mapped the minimal region ofNup358 necessary to compensate the import defect. On the other hand, we could confirm therequirement of CRM1 in nuclear targeting of AdV capsids and identified and additional role inmediating AdV capsid disassembly. This work helps to understand the strategy used by AdV todeliver their genome and gives insight about how viruses hijack the cellular transport machinery fortheir own benefit.
... Intracellular trafficking of HPK follows a similar pattern as that of wild-type PB ( Figure 3A), which rapidly transits toward the nuclear periphery after cell entry (52,58), similar to the whole virus (59)(60)(61). Subcellular fractionation shows substantial entry of HPK into cytoplasmic and cytoskeletal compartments after uptake into HER3-expressing MDA-MB-435 cells ( Figure 3A; relative cell surface HER3 levels shown in Supplementary Figure S2). These findings compare favorably with those for the wild-type PB, for which a considerable proportion is internalized to cytoskeletal and nuclear compartments (similarly to the trafficking of whole Ad) (59-61) that are otherwise inaccessible without endosomal disruption, while a small proportion was retained in the membrane fraction ( Figure 3A). ...
... First, the basic foundation upon which HPK was built is the Ad5 capsid PB, which was functionally shown to bear cell-penetrating properties (33,52,58) despite an unknown mechanism of membrane destabilization. Intracellular trafficking studies of subgroup C viruses, including Ad2 and Ad5 which share high sequence identity (67), have shown that the viral particles attach to dynein motors after endosomal escape and are ferried along intact microtubules as naked particles toward the nuclear periphery (61). These particles appear as discrete puncta undergoing minus-end directed movement in fluorescence-based imaging studies (68,69). ...
RNA interference represents a potent intervention for cancer treatment but requires a robust delivery agent for transporting gene-modulating molecules, such as small interfering RNAs (siRNAs). Although numerous molecular approaches for siRNA delivery are adequate in vitro, delivery to therapeutic targets in vivo is limited by payload integrity, cell targeting, efficient cell uptake, and membrane penetration. We constructed nonviral biomaterials to transport small nucleic acids to cell targets, including tumor cells, on the basis of the self-assembling and cell-penetrating activities of the adenovirus capsid penton base. Our recombinant penton base chimera contains polypeptide domains designed for noncovalent assembly with anionic molecules and tumor homing. Here, structural modeling, molecular dynamics simulations, and functional assays suggest that it forms pentameric units resembling viral capsomeres that assemble into larger capsid-like structures when combined with siRNA cargo. Pentamerization forms a barrel lined with charged residues mediating pH-responsive dissociation and exposing masked domains, providing insight on the endosomolytic mechanism. The therapeutic impact was examined on tumors expressing high levels of HER3/ErbB3 that are resistant to clinical inhibitors. Our findings suggest that our construct may utilize ligand mimicry to avoid host attack and target the siRNA to HER3+ tumors by forming multivalent capsid-like structures.
... Adenoviral trafficking is reported to require intact microtubules (Leopold et al., 2000), and mutations in dynamin 2 increase acetylation and accumulation of stable microtubules (Tanabe and Takei, 2009). As our results above indicated an increase in viral gene expression upon dynamin 2 knockdown, we further investigated whether changes in microtubule stability contributed to increased viral trafficking. ...
... Earlier reports indicated that various viruses use microtubules to access the nucleus (Leopold et al., 1998(Leopold et al., , 2000Nakano and Greber, 2000;Suomalainen et al., 1999), specifically that stable, non-dynamic microtubules enhance nuclear-directed viral trafficking (Mabit et al., 2002;Naranatt et al., 2005;Suomalainen et al., 2001). Based on the data above, we questioned whether the viruses that enter cells knocked down for dynamin 2 are contained within endosomes or found free in the cytosol. ...
The large GTPase dynamin 2 controls both endosomal fission and microtubule acetylation. Here we report that dynamin 2 alters microtubules and regulates the trafficking of human adenovirus type 37. Dynamin 2 knockdown by siRNA in infected cells resulted in accumulation of acetylated tubulin, repositioning of microtubule organizing centers (MTOCs) closer to cell nuclei, increased virus in the cytosol (with a compensatory decrease in endosomal virus), reduced proinflammatory cytokine induction, and increased binding of virus to the nucleoporin, Nup358. These events led to increased viral DNA nuclear entry and viral replication. Overexpression of dynamin 2 generated opposite effects. Therefore, dynamin 2 inhibits adenovirus replication and promotes innate immune responses by the infected cell. MTOC transposition in dynamin 2 knockdown promotes a closer association with nuclear pore complexes to facilitate viral DNA delivery. Dynamin 2 plays a key role in adenoviral trafficking and influences host responses to infection.
... Adenovirus enters target cells by receptor-mediated endocytosis [14]. Following endosomal escape, Ad particles travel along microtubules (MT), and then dock at nuclear pore complexes (NPC) to deliver their DNA genome into the nucleus [4,15,16]. In enucleated cells or those treated with the nuclear export inhibitor leptomycin B, capsids bypass the nucleus and accumulate in the vicinity of the centrosome [17,18]. ...
... In enucleated cells or those treated with the nuclear export inhibitor leptomycin B, capsids bypass the nucleus and accumulate in the vicinity of the centrosome [17,18]. Transport to the cell center involves the MT minus end-directed motor protein cytoplasmic dynein, and can be inhibited by microtubule-destabilizing agents or blocking dynein or dynactin function using dominant negative cDNAs, RNAi, or acutely injected function-blocking antibodies and inhibitory fragments directed at dynein subunits [4,5,15,19,20]. ...
Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based “assisted-diffusion” behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a behavior of potential importance for physiological cargoes as well.
... The exact means by which the penton base disrupts the endosomal membrane integrity has long been unknown, although recent studies have begun to uncover this elusive mechanism as discussed later in this review. Studies on Ad5 intracellular trafficking show that postendosomal capsids migrate toward the nucleus by way of the microtubules, specifically by interacting with the cytoplasmic dynein complexes that normally transport vesiculated cargo within the cell [65,66]. Importantly, soluble recombinant Ad5 penton base is capable of microtubule-mediated trafficking through the cytoplasm to reach the cell nucleus in the absence of the rest of the virus and in a similar manner to that of the whole adenovirus ( Figure 2d) [23,61]. ...
Macromolecular therapeutics such as nucleic acids, peptides, and proteins have the potential to overcome treatment barriers for cancer. For example, nucleic acid or peptide biologics may offer an alternative strategy for attacking otherwise undruggable therapeutic targets such as transcription factors and similar oncologic drivers. Delivery of biological therapeutics into tumor cells requires a robust system of cell penetration to access therapeutic targets within the cell interior. A highly effective means of accomplishing this may be borrowed from cell-penetrating pathogens such as viruses. In particular, the cell entry function of the adenovirus penton base capsid protein has been effective at penetrating tumor cells for the intracellular deposition of macromolecular therapies and membrane-impermeable drugs. Here, we provide an overview describing the evolution of tumor-targeted penton-base-derived nano-capsids as a framework for discussing the requirements for overcoming key barriers to macromolecular delivery. The development and pre-clinical testing of these proteins for therapeutic delivery has begun to also uncover the elusive mechanism underlying the membrane-penetrating function of the penton base. An understanding of this mechanism may unlock the potential for macromolecular therapeutics to be effectively delivered into cancer cells and to provide a treatment option for tumors resisting current clinical therapies.
... This process gives Ads enough time to access the cytoplasm to engage in motor binding for microtubule-directed transport to the nucleus to deliver their genome (15)(16)(17)(18). Despite the partial disassembly required for endosomal escape, Ads remain as physical entities during cytoplasmic transport, and several studies have shown that particles enrich at the level of the microtubule organizing center (MTOC) prior to their accumulation at the nuclear envelope (NE) (19)(20)(21)(22). There, Ad particles dock via hexon at the nuclear pore complex (NPC) by binding Nup214 (23,24), a nucleoporin that faces the cytoplasmic side of the NPC (25). ...
After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor.
Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly, both in interphase and in mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export.
IMPORTANCE
A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export-competent but deficient in viral capsid disassembly, both in interphase and in mitotic cells.
... Once Ads have reached the cytosol, they engage with dynein motors and use retrograde transport along microtubules to reach the microtubule organizing center [115][116][117]. Motor binding is probably mediated by hexon [115] and may require acidic priming [118]. ...
Adenovirus vector-based genetic vaccines have emerged as a powerful strategy against the SARS-CoV-2 health crisis. This success is not unexpected because adenoviruses combine many desirable features of a genetic vaccine. They are highly immunogenic and have a low and well characterized pathogenic profile paired with technological approachability. Ongoing efforts to improve adenovirus-vaccine vectors include the use of rare serotypes and non-human adenoviruses. In this review, we focus on the viral capsid and how the choice of genotypes influences the uptake and subsequent subcellular sorting. We describe how understanding capsid properties, such as stability during the entry process, can change the fate of the entering particles and how this translates into differences in immunity outcomes. We discuss in detail how mutating the membrane lytic capsid protein VI affects species C viruses’ post-entry sorting and briefly discuss if such approaches could have a wider implication in vaccine and/or vector development.
... Then, the Ads enter cells via endocytosis mediated by the interaction between the integrins on the cell surface and an arginineglycine-aspartate motif located in the penton base, followed by escape to the cytosol [6,7]. After the viral genomes are transported along the microtubules in a dynein-dependent manner, they are translocated to the nucleus [8,9], where viral DNA replication is activated by the early region proteins, such as E1A and E1B [10,11]. After the formation of progeny Ad particles in the nucleus, infected cells are lysed by E3-11.6 K protein, followed by particle spread to uninfected cells via cell-free transmission [12,13]. ...
Human adenoviruses (Ads), common pathogens that cause upper respiratory and gastrointestinal infections, are blocked by neutralizing antibodies (nAbs). However, Ads are not fully eliminated even in hosts with nAbs. In this study, we assessed the infectivity of progeny Ad serotype 5 (Ad5) in the presence of nAb. The infectivity of Ad5 was evaluated according to the expression of the Ad genome and reporter gene. Infection by wild-type Ad5 and Ad5 vector continued to increase until 3 days after infection even in the presence of nAb. We established an assay for determining the infection levels of progeny Ad5 using a sorting system with magnetic beads and observed little difference in progeny Ad5 counts in the presence and absence of nAb 1 day after infection. Moreover, progeny Ad5 in the presence of nAb more effectively infected coxsackievirus and adenovirus receptor (CAR)-positive cells than CAR-negative cells. We investigated the function of fiber proteins, which are the binding partners of CAR, during secondary infection, observing that fibre proteins spread from infected cells to adjacent cells in a CAR-dependent manner. In conclusion, this study revealed that progeny Ad5 could infect cells even in the presence of nAb, differing from the common features of the Ad5 infection cycle. Our findings may be useful for developing new therapeutic agents against Ad infection.
... Microtubules (MTs) are dynamic cytoskeletal components that nucleate predominantly from the microtubule organizing center (MTOC), often synonymous with the nuclear-proximal centrosome. By hijacking MT tracks, the virus scales most of the distance to its nuclear destination (9). ...
Significance
Adenoviruses (AdVs) are DNA viruses that can cause severe respiratory illness in humans. A better understanding of the complex ways AdVs utilize cellular processes in service of their replication is critical to the development of new therapies to counter viral infection and disease. Using a genome-wide screen, we identified a cellular protein Mindbomb 1 (MIB1) required for AdV infection and show that it functions in the AdV entry process to mediate release of the viral genome into the nucleus. We further show that MIB1, as an E3 ubiquitin ligase, mediates AdV capsid disassembly and genome release through ubiquitination of a target protein(s), demonstrating the importance of this rapid posttranslational modification to virus infection.
... - 2 0 -After escaping from the endosome, Ad enters the cytoplasm where it associates with microtubules and is shuttled to the microtubule organizing center prior to translocation to the nucleus (Bailey, Crystal et al. 2003). The virus moves through the cytosol at speeds of up to 2 micrometers (pm )/second (sec), driven by dynein motors along the microtubules (Leopold, Ferris et al. 1998;Leopold, Kreitzer et al. 2000;Kelkar, Pfister et al. 2004). Once Ad reaches the nuclear envelope, it docks with the nuclear pore complex and the final uncoating of the virus occurs Chardonnet and Dales 1972;Greber, Suomalainen et al. 1997;Trotman, Mosberger et al. 2001). ...
Recombinant adenovirus (Ad) is a powerful tool in gene therapy. However, the ability to deliver Ad to target tissues and cells following systemic administration is limited due to rapid clearance from blood circulation by the mononuclear phagocyte system (MPS), leading to transfection of non-target tissues (primarily liver), vector related toxicity and immunogenicity. The aim of this work was to engineer synthetic lipid envelopes around adenovirus in order to develop a novel gene therapy vector based on a hybrid viral/non-viral vector platform that would address some of the limitations of Ad. The interaction between the virions and phospholipid bilayers was systematically studied in order to develop a protocol for constructing this vector by using self-assembly principles without any alteration of the Ad genome or application of surface conjugation chemistry The hypothesis that envelopment of Ad using different lipid bilayers would produce nanoparticles with characteristics determined by the type of lipid used was tested using a variety of structural, physicochemical, and biological assays. Vector physicochemical characteristics such as size, degree of aggregation, stability in suspension, and surface charge were linked to biological function in vitro and in vivo. The results indicated several key parameters toward the development of stable, non-flocculated suspensions of lipid enveloped Ad, and how such characteristics affected the gene transfer efficiency, organ distribution, and pharmacokinetics of the resulting vectors. Further, these results demonstrated that it was possible to overcome problems of inherent viral tissue affinities and to reduce immunogenicity by introduction of the synthetic envelope. Next, the lipid components of the envelope were altered to optimize the vector for cancer therapy. Inclusion of stealth components like poly(ethylene glycol) (PEG)-lipids in the envelope extended blood circulation time. It was also found that enveloped Ad vectors could be tailored to preferentially accumulate in tumor tissue with little interaction with non-target tissue in vivo. Further, these vectors were demonstrated to possess enhanced tumor-penetration capability in vitro. Finally, the possibility of enhancing the therapeutic effect and reducing the toxicity of conditionally replication competent adenovirus (CRAd) used for cancer therapy was evaluated using both in vitro and in vivo models of human tumors. Although no improvements were observed in cancer therapy, the enveloped CRAd was far less toxic than the unmodified virus. This may lead to the development of cancer targeting vectors with low systemic toxicity, which will greatly broaden the horizon for oncolytic virotherapy of cancer. In summary, the studies in this thesis provide a new paradigm for modification of non-enveloped virions (such as Ad) by application of self-assembly principles and have contributed to a greater understanding of the importance of characterizing and manipulating Ad's physicochemical characteristics to enhance its biological activity. As such, this thesis provides the basis for development of a novel vector platform that may be used for the systemic treatment of primary or broadly disseminated cancer.
... The intracellular domain of CAR plays a critical role in this by recruiting the endocytic machinery and influencing subsequent intracellular AdV trafficking (Loustalot et al., 2015). AdVs are then transported towards the nucleus by cytoplasmic dyneinmediated trafficking along with the microtubule network (Kelkar et al., 2004), impairments in which drastically disrupt this nuclear targeting (Suomalainen et al., 1999;Leopold et al., 2000). The AdV capsid directly interacts with cytoplasmic dynein via hexon capsomeres (Bremner et al., 2009), suggesting that in non-neuronal cells AdVs are transported as ''naked particles'' rather than in membrane-bound organelles (e.g., endosomes; Scherer et al., 2020). ...
Virus-mediated gene therapy has the potential to deliver exogenous genetic material into specific cell types to promote survival and counteract disease. This is particularly enticing for neuronal conditions, as the nervous system is renowned for its intransigence to therapeutic targeting. Administration of gene therapy viruses into skeletal muscle, where distal terminals of motor and sensory neurons reside, has been shown to result in extensive transduction of cells within the spinal cord, brainstem, and sensory ganglia. This route is minimally invasive and therefore clinically relevant for gene therapy targeting to peripheral nerve soma. For successful transgene expression, viruses administered into muscle must undergo a series of processes, including host cell interaction and internalization, intracellular sorting, long-range retrograde axonal transport, endosomal liberation, and nuclear import. In this review article, we outline key characteristics of major gene therapy viruses—adenovirus, adeno-associated virus (AAV), and lentivirus—and summarize the mechanisms regulating important steps in the virus journey from binding at peripheral nerve terminals to nuclear delivery. Additionally, we describe how neuropathology can negatively influence these pathways, and conclude by discussing opportunities to optimize the intramuscular administration route to maximize gene delivery and thus therapeutic potential.
... Upon endosomal escape of viruses, reverse signaling from the host molecular machinery to the virus facilitates the release of the viral genome [29]. In nonacidic early-stage endosomes and macropinosomes containing incoming virions, since damaged adenoviruses expose protein VI to the plasma membrane upon cell entry, causing the membrane to be leaky [25], damaged virions in endosomes could be primed for endosomal escape via the leakage of membranes in concert with the The viral particles released from endosomes are transported toward the minus ends of microtubules near the centrosome and the nucleus across the densely packed cytosol [73]. Although the complexity of microtubule-dependent virion movements that are executed by contra-directional microtubule motors, which consist of plus-end-directed kinesin Kif5B and KLC1/2 and minus-end-directed dynein/dynamin, still remain to be elucidated [74], a stochastic model in which the directionality can be determined with certainty was proposed [75] that well explained the movement of peroxisomes in the cytosol [76]. ...
Mitochondrial transfer has been recognized to play a role in a variety of processes, ranging from fertilization to cancer and neurodegenerative diseases as well as mammalian horizontal gene transfer. It is achieved through either exogeneous or intercellular mitochondrial transfer. From the viewpoint of evolution, exogeneous mitochondrial transfer is quite akin to the initial process of symbiosis between α-protobacterium and archaea, although the progeny have developed more sophisticated machinery to engulf environmental materials, including nutrients, bacteria, and viruses. A molecular-based knowledge of endocytosis, including macropinocytosis and endosomal escape involving bacteria and viruses, could provide mechanistic insights into exogeneous mitochondrial transfer. We focus on exogeneous mitochondrial transfer in this review to facilitate the clinical development of the use of isolated mitochondria to treat various pathological conditions. Several kinds of novel procedures to enhance exogeneous mitochondrial transfer have been developed and are summarized in this review.
... This results in loss of the fiber protein and exposure of the membrane lytic protein VI, which lyses the endosomal membrane. Upon entry into the cytosol, hexon recruits dynein and the capsid moves toward the nucleus (105)(106)(107)(108)(109). During this process, the capsid continues to progressively disassemble into a more metastable state that ultimately serves to deliver the viral genome into the nucleus. ...
Tripartite motif containing-21 (TRIM21) is a cytosolic ubiquitin ligase and antibody receptor that provides a last line of defense against invading viruses. It does so by acting as a sensor that intercepts antibody-coated viruses that have evaded extracellular neutralization and breached the cell membrane. Upon engagement of the Fc of antibodies bound to viruses, TRIM21 triggers a coordinated effector and signaling response that prevents viral replication while at the same time inducing an anti-viral cellular state. This dual effector function is tightly regulated by auto-ubiquitination and phosphorylation. Therapeutically, TRIM21 has been shown to be detrimental in adenovirus based gene therapy, while it may be favorably utilized to prevent tau aggregation in neurodegenerative disorders. In addition, TRIM21 may synergize with the complement system to block viral replication as well as transgene expression. TRIM21 can also be utilized as a research tool to deplete specific proteins in cells and zebrafish embryos. Here, we review our current biological understanding of TRIM21 in light of its versatile functions.
... Once Ad5 has escaped the endosome, hexon recruits dynein and travels along microtubules to the nucleus, where it interacts with the nuclear pore complex to deliver the viral genome into the nucleus (Bremner et al., 2009;Leopold et al., 2000;Suomalainen et al., 1999;Cassany et al., 2015;Trotman et al., 2001). Given that complement inhibits viral entry into the cytosol, we hypothesized that this should result in fewer viruses reaching the nuclear membrane. ...
The complement system is vital for anti-microbial defense. In the classical pathway, pathogen-bound antibody recruits the C1 complex (C1qC1r 2 C1s 2 ) that initiates a cleavage cascade involving C2, C3, C4, and C5 and triggering microbial clearance. We demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components. C4 inhibits human adenovirus infection by directly inactivating the virus capsid. Rapid C4 activation and capsid deposition of cleaved C4b are catalyzed by antibodies via the classical pathway. Capsid-deposited C4b neutralizes infection independent of C2 and C3 but requires C1q antibody engagement. C4b inhibits capsid disassembly, preventing endosomal escape and cytosolic access. C4-deficient mice exhibit heightened viral burdens. Additionally, complement synergizes with the Fc receptor TRIM21 to block transduction by an adenovirus gene therapy vector but is partially restored by Fab virus shielding. These results suggest that the complement system could be altered to prevent virus infection and enhance virus gene therapy efficacy. The complement system is pivotal to the humoral immune response. Bottermann et al. demonstrate that complement proteins C1 and C4 possess potent antiviral activity independent of downstream components. C4b is deposited on the viral capsid, preventing capsid disassembly and virus entry into the cytosol, a prerequisite for productive virus infection.
... During this period, viruses need to travel along cytoskeleton to reach a specific site for genome release after entering the cell, or travel inside endocytic or exocytic vesicles 8 . During viral transport in the cytoplasm viral proteins interact directly with dynein 47 or kinesin 48 motors. Since viruses widely exploit diverse cellular mechanisms for trans- port, the virus motion modes and the movement speeds may be more complicated during this period for PEDV. ...
In order to study the infection mechanism of porcine epidemic diarrhea virus (PEDV), which causes porcine epidemic diarrhea, a highly contagious enteric disease, we combined quantum dot labeled method, which could hold intact infectivity of the labeled viruses to the largest extent, with the single particle tracking technique to dynamically and globally visualize the transport behaviors of PEDVs in live Vero cells. Our results were the first time to uncover the dynamic characteristics of PEDVs moving along the microtubules in the host cells. It is found that PEDVs kept restricted motion mode with a relatively stable speed in the cell membrane region; while performed a slow-fast-slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule organizing center region. In addition, the return movements of small amount of PEDVs were also observed in the live cells. Collectively, our work is crucial for understanding the movement mechanisms of PEDV in the live cells, and the proposed work also provided important references for further analysis and study on the infection mechanism of PEDVs.
... The partially uncoated HAdV capsid is then released into the cytoplasm and transported to the nuclear pore complex by the microtubular network (Smith et al., 2008;Wiethoff et al., 2005). Moreover, HAdV associates with microtubules following endosome penetration by the presence of microtubule-associated proteins (MAPs) and the block of this association can prevent HAdV infection (Bailey et al., 2003;Kelkar et al., 2004;Leopold et al., 2000;Luftig and Weihing, 1975;Smith et al., 2008;Suomalainen et al., 1999Suomalainen et al., , 2001. One example of the functional relevance of this process is the anti-hexon neutralizing antibody 9C12, a monoclonal antibody that blocks HAdV infection at the microtubule-dependent transport stage (Smith et al., 2008). ...
The repurposing of drugs approved by the regulatory agencies for other indications is emerging as a valuable alternative for the development of new antimicrobial therapies, involving lower risks and costs than the de novo development of novel antimicrobial drugs. Adenovirus infections have showed a steady increment in recent years, with a high clinical impact in both immunosuppressed and immunocompetent patients. In this context, the lack of a specific drug to treat these infections supports the search for new therapeutic alternatives. In this study, we examined the anti-HAdV properties of mifepristone, a commercially available synthetic steroid drug. Mifepristone showed significant in vitro anti-HAdV activity at low micromolar concentrations with little cytotoxicity. Our mechanistic assays suggest that this drug could affect the microtubule transport, interfering with the entry of the virus into the nucleus and therefore inhibiting HAdV infection.
... There is loss of peripentonal hexons, pIIIa, pVIII, and pIX, as well as the release of pVI, a minor capsid protein required for disruption of the endosomal membrane 18,37,[45][46][47] and facilitation of nuclear importation through movement in a microtubule-and dynein-dependent mechanism and dock at the nuclear pore complex, a proteinaceous channel that mediates movement of the nucleocapsid viral genome into the nucleus, where viral replication takes place. 18,37,48,49 The replication cycle of AdV consists of an early stage and a late stage. 50 The early stage of adenoviral replication is characterized by the generation of adenoviral E1A protein (encoded by the E1A gene) that transactivates other early genes E1B, E2, E3, and E4, which play important roles in the early stage of the AdV replication cycle. ...
Human adenovirus (HAdV) is a ubiquitous virus that infects the mucosa of the eye. It is the most common cause of infectious conjunctivitis worldwide, affecting people of all ages and demographics. Pharyngoconjunctival fever outbreak is due to HAdV types 3, 4, and 7, whereas outbreaks of epidemic keratoconjunctivitis are usually caused by HAdV types 8, 19, 37, and 54. Primary cellular receptors, such as CAR, CD46, and sialic acid interact with fiber-knob protein to mediate adenoviral attachment to the host cell, whereas adenoviral penton base–integrin interaction mediates internalization of adenovirus. Type 1 immunoresponse to adenoviral ocular infection involves both innate immunity mediated by natural killer cells and type 1 interferon, as well as adaptive immunity mediated mainly by CD8 T cells. The resulting ocular manifestations are widely variable, with pharyngoconjunctival fever being the most common, manifesting clinically with fever, pharyngitis, and follicular conjunctivitis. Epidemic keratoconjunctivitis, however, is the severest form, with additional involvement of the cornea leading to development of subepithelial infiltrates. Because there is currently no US Food and Drug Administration-approved treatment for adenoviral ocular infection, current management is palliative. The presence of sight-threatening complications following ocular adenoviral infection warrants the necessity for developing antiadenoviral therapy with enhanced therapeutic index. Future trends that focus on adenoviral pathogenesis, including adenoviral protein, which utilize host receptors to promote infection, could be potential therapeutic targets, yielding shorter active disease duration and reduced disease burden.
... In the endosome, the pentons are released together with some internal components including protein VI, which has been shown to induce a pH-independent disruption of the endosomal membrane [20,21]. Then the partially uncoated capsid traffics to the nuclear pore complex [22,23] where the final uncoating takes place, allowing the viral genome to enter the nucleus [24]. Nanoindentation by atomic force microscopy (AFM) is an emergent technique to characterize the mechanical properties of artificial as well as natural nano-sized structures [25][26][27][28]. ...
Viruses are extensively studied as vectors for vaccine applications and gene therapies. For these applications, understanding the material properties of viruses is crucial for creating optimal functionality. Using atomic force microscopy (AFM) nanoindentation, we studied the mechanical properties of human adenovirus type 5 with the fiber of type 35 (Ad5F35) and compared it to viral capsids with a single point mutation in the protein VI precursor protein (pVI-S28C). Surprisingly, the pVI-S28C mutant turned out to be twice as stiff as the Ad5F35 capsids. We suggest that this major increase in strength is the result of the DNA crosslinking activity of precursor protein VII, as this protein was detected in the pVI-S28C mutant capsids. The infectivity was similar for both capsids, indicating that mutation did not affect the ability of protein VI to lyse the endosomal membrane. This study highlights that it is possible to increase the mechanical stability of a capsid even with a single point mutation while not affecting the viral life cycle. Such insight can help enable the development of more stable vectors for therapeutic applications.
Electronic supplementary material
The online version of this article (10.1007/s10867-017-9479-y) contains supplementary material, which is available to authorized users.
... They enter epithelial cells by receptor-mediated endocytosis (Meier et al., 2002;Wolfrum and Greber, 2013), disrupt the endosomal membrane, and escape as membrane- free particles into the cytosol ( Luisoni et al., 2015;Moyer et al., 2011). The transport of adenovirus towards the microtubule-organizing center (MTOC, often located near the nucleus) depends on the dynein/dynactin motor complex (Bremner et al., 2009;Engelke et al., 2011;Kelkar et al., 2006;Kelkar et al., 2004;Leopold et al., 2000;Suomalainen et al., 1999). It is enhanced by cell signaling, such as p38/MAPK and protein kinase A (PKA) pathways, which are activated by incoming virions (Wolfrum and Greber, 2013), together with the ERK/MAPK pathway (Bruder and Kovesdi, 1997). ...
Transport of large cargo through the cytoplasm requires motor proteins and polarized filaments. Viruses that replicate in the nucleus of post-mitotic cells use microtubules and the dynein/dynactin motor to traffic to the nuclear membrane, and deliver their genome through nuclear pore complexes (NPCs) into the nucleus. How virus particles (virions) or cellular cargo are transferred from microtubules to the NPC is unknown. Here, we analyzed trafficking of incoming cytoplasmic adenoviruses by single particle tracking and super-resolution microscopy. We provide evidence for a regulatory role of CRM1/XPO1 (chromosome-region-maintenance-1, exportin-1) in juxta-nuclear microtubule-dependent adenovirus transport. Leptomycin B (LMB) abolishes nuclear targeting of adenovirus. It binds to CRM1, precludes CRM1-cargo binding and blocks signal-dependent nuclear export. LMB-inhibited CRM1 did not compete with adenovirus for binding to the nucleoporin Nup214 at the NPC. Instead, CRM1 inhibition selectively enhanced virion association with microtubules, and boosted virion motions on microtubules less than about 2 µm from the nuclear membrane. The data show that the nucleus provides positional information for incoming virions to detach from microtubules, engage a slower microtubule-independent motility to the NPC and enhance infection.
... Protein VI then proceeds to lyse the endosome to facilitate viral escape (102). The partially uncoated virus is then transported to the nuclear pores by microtubules (103)(104)(105). The virus then associates with the nuclear pore complex through interaction with the hexon protein and the final stages of virus uncoating occur ( Figure 6) (106)(107)(108). ...
Adenoviruses are amongst the most common pathogens infecting animals and humans. Due to their simple structure and ability to be easily modified, they are widely used as tools for studying cellular processes, or in clinical applications as gene-delivery vectors.
Antibodies directed against viral proteins serve as an important diagnostic tool and help to elucidate infection mechanisms and infection pathways in cells and organisms. Virus-specific antibodies are usually generated by using purified viruses or subviral components as antigens. However, not all viruses can be cultured or produced in sufficient amounts for them to be used as antigens for antibody production. Here we describe a method for cloning, heterologous expression and purification of the hypervariable region of the adenoviral hexon protein, which can be used as a potent antigen for generating antibodies against the virus of origin.
Using this method, we were able to generate polyclonal antibodies against the mouse adenovirus 1 hexon protein. The serum is able to detect the full length hexon protein in cell lysates or purified virus stocks. Additionally, we produced hexon fragments of the human adenovirus 5 and 3, which were characterized using an antibody blocking assay in cells infected with a luciferase expressing virus. The method described here could potentially be applied for a variety of adenoviruses that are difficult to amplify in cell culture, to generate novel tools for diagnostics and research.
Viruses evolve very rapidly and their genome accumulates numerous mutations during each replication. Because of this, every time a virus is propagated in cell culture, there is a possibility that the amplified virus will be carrying additional mutations that were not present in the original virus. Therefore, it is necessary to regularly sequence the genomes of the viruses that are worked with, to assure that there are no mutations in genes that could affect the phenotype of the virus. To this end, genomes of several adenoviruses that are commonly used in our group were sequenced using next-generation sequencing techniques. Additionally, we sequenced the genome of a variant strain of human adenovirus 40 F (HoviX) to compare it to the reference strain (Dugan) and see if there are single nucleotide variations present that could affect the virus.
... The replication cycle of DENV has been extensively studied with particular regard to early events such as binding, fusion, uncoating, and intracellular transport of viral proteins [28,49,50]. On the other hand, the relationships between the replication of DENV and the cytoskeleton host cell are not fully characterized, although data in some literature, concerning influenza and other viruses, support their existence [13,[51][52][53][54][55][56]. ...
... Newly emerging information indicates that transport of the virus to the nuclear pore occurs via microtubules and dynein motors [90][91][92][93]. The final "undressing" of the adenovirus capsid at the nuclear pore complex is necessitated by the observation that macromolecular complexes of greater than 40 nm in diameter are unable to enter the nucleus by simple passive diffusion [94]. ...
Human adenoviruses are large (150 MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause acute respiratory, gastrointestinal and ocular infections. Despite these disease associations, adenovirus has aided basic and clinical research efforts through studies of its association with cells and as a target of host antiviral responses. This review highlights the knowledge of adenovirus disassembly and nuclear transport gleaned from structural, biophysical and functional analyses of adenovirus interactions with soluble and membrane-associated host molecules.
... Several studies have shown that an intact microtubule network and motor proteins such as dynein are necessary for transfected naked DNA and some viruses to traverse the cytoplasm and reach the nucleus [102,266,268,269]. When the microtubule network was disassembled by treatment with nocodazole, movement of DNA toward the nucleus was largely abolished [102,266]. ...
Cells are the structural and functional unit of all living organisms and exhibit fundamental properties of life. Cells are surrounded by the cell membrane and subdivided into various compartments. Pulsed electric fields (PEFs) exert profound effects on cells by interacting with the cell membrane and other cellular components. This chapter describes the biological effects of PEF at cellular and subcellular levels. First, this chapter begins with the overview of cell exposure to PEF from a biophysical point of view. Second, the interaction of PEF with biological membranes, membrane pore formation, and their physiological significance is described from multifaceted standpoints. Next, this chapter explains subcellular events induced by PEF, including the effect on cytoskeleton and signal transduction. Lastly, detailed description on irreversible electroporation and cell death by PEF is provided. The topics covered in this chapter serve as the basis for the applications of PEF in medicine, environmental science, and food and biomass processing.
... Viruses have evolved multiple ways of hijacking the cellular transport machinery to propel themselves through the cell, from their site of entry to their sites of replication and egress (2)(3)(4)(5)(6)(7). In particular, viruses that replicate in the nucleus, such as adenoviruses (8,9), retroviruses (10,11), and herpes viruses (12,13), use the MT network and associated motors to traffic within the cytoplasm either from the cell periphery to the center of the cell (dynein) or in the reverse direction (kinesins). The importance of molecular motors in viral transport is well established and has been reviewed elsewhere (14,15). ...
Viruses are entirely dependent on their ability to infect a host cell in order to replicate. To reach their site of replication as rapidly and efficiently as possible following cell entry, many have evolved elaborate mechanisms to hijack the cellular transport machinery to propel themselves across the cytoplasm. Long-range movements have been shown to involve motor proteins along microtubules (MTs) and direct interactions between viral proteins and dynein and/or kinesin motors have been well described. Although less well-characterized, it is also becoming increasingly clear that non-motile microtubule-associated proteins (MAPs), including structural MAPs of the MAP1 and MAP2 families, and microtubule plus-end tracking proteins (+TIPs), can also promote viral trafficking in infected cells, by mediating interaction of viruses with filaments and/or motor proteins, and modulating filament stability. Here we review our current knowledge on non-motile MAPs, their role in the regulation of cytoskeletal dynamics and in viral trafficking during the early steps of infection.
... Knob . Die am CAR gebundenen Ad werden über die als Vitronektinund FN-Rezeptoren bekannten Integrine αvβ3 und αvβ5 internalisiert und anschließend über mikrotubuläre Translokationssysteme zum Zellkern geschleust (Johansson et al., 1997;Schvartz et al., 1999;Wickham et al., 1993;Leopold et al. 2000). Interessanterweise ist die ...
The coxsackievirus-adenovirus receptor (CAR), a transmembrane protein of the Immunoglobulin
superfamily (IgSF) composed of two extracellular Ig domains (D1 and D2), is
strongly expressed in the developing central nervous system. Based on its localization it is
possibly involved in the formation of synapses or axonal outgrowth. Beside its role as a cellular
virus receptor the cell biological function in the nervous system remains to be revealed.
Consistently, antibodies to CAR block the attachment of neurons on glycoproteins of the extracellular
matrix (ECM) or disturb neurite extension on basal laminae preparations in vitro.
In this thesis binding studies showed that fibronectin (FN), laminin-1, fibulin-1, tenascin-
R and agrin are extracellular interaction partners of CAR. Most likely through these interactions
CAR stimulates processes like cell-adhesion and neurite extension. Concerning FN
the interaction could be mapped to a fragment bearing the second heparin-binding-domain
(FN-40-kDa), which is different from the main integrin binding segment, and to the second Ig
domain (D2) of CAR. This FN-fragment has a neurite-stimulating activity in cell-culture
which is abolished on neurons from CAR-deficient mice or when CAR is blocked by antibodies.
Beside this heterophilic binding CAR reveals homophilic binding as well. For example,
the homotypic interaction of CAR-deficient neurons on FN-40-kDa is disturbed. Furthermore,
the aggregation of CAR-transfected NIH 3T3 and CHO cells is stimulated compared
to their parental cells. As known for several IgSF-members the extracellular part of
CAR binds to itself which is favored when it is N-glycosylated. However, high concentrations
of bacterially expressed extracellular CAR form also dimers, which is obeserved in the cristallographic
structure, where a D1 D1 interaction is visible. Again through binding studies, evidence
is given that alternatively to the already described D1 D1 interaction a trans D1 D2
interaction takes place with a 20-fold increased affinity.
The intracellular segment of CAR is found to bind to the actin-cytoskeleton by interacting
directly with α-actinin and profilin-1.
It can be concluded that CAR acts, like integrins and syndecans, as a linker between
the ECM or itself on adjacent cells and the actin-cytoskeleton.
... Consistent with our findings, many other viruses, including Ad, HSV, and HIV, have also been shown to move on MTs to the MTOC region and associated with MT-MTOC. 10,14,39,40,[57][58][59][60][61][62] Furthermore, Fig. 1E shows that only a small portion of viral particles co-localized with the dispersed Golgi cisternae, which are released from highly packed MTOC region upon MT-MTOC disruption. This suggests that fewer particles physically localize in the Golgi apparatus. ...
Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After Nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) post virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting the MT-MTOC in brain, liver and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at the MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion.
As of 10 May 2022, at least 450 cases of pediatric patients with acute hepatitis of unknown cause have been reported worldwide. Human adenoviruses (HAdVs) have been detected in at least 74 cases, including the F type HAdV41 in 18 cases, which indicates that adenoviruses may be associated with this mysterious childhood hepatitis, although other infectious agents or environmental factors cannot be excluded. In this review, we provide a brief introduction of the basic features of HAdVs and describe diseases caused by different HAdVs in humans, aiming to help understand the biology and potential risk of HAdVs and cope with the outbreak of acute child hepatitis.
The cytoskeleton is an essential component of the cell and it is involved in multiple physiological functions, including intracellular organization and transport. It is composed of three main families of proteinaceous filaments; microtubules, actin filaments and intermediate filaments, and their accessory proteins. Motor proteins, which comprise the dynein, kinesin, and myosin superfamilies, are a remarkable group of accessory proteins that mainly mediate the intracellular transport of cargoes along the cytoskeleton. Like other cellular structures and pathways, viruses can exploit the cytoskeleton to promote different steps of their life cycle through associations with motor proteins. The complexity of the cytoskeleton and the differences among viruses, however, has led to a wide diversity of interactions, which in most cases remain poorly understood. Unveiling the details of these interactions is necessary not only for a better comprehension of specific infections, but may also reveal new potential drug targets to fight dreadful diseases such as the rabies disease and the acquired immunodeficiency syndrome (AIDS). In this review, we describe a few examples of the mechanisms that some human viruses, i.e., rabies virus, adenovirus, herpes simplex virus, human immunodeficiency virus, influenza A virus and papillomavirus, have developed to hijack dyneins, kinesins and myosins. This article is protected by copyright. All rights reserved. Microtubules, actin filaments and intermediate filaments are central elements of the cell's cytoskeleton. Motor proteins such as dyneins, kinesins and myosins allow directional intracellular transport of cargoes along these cytoskeleton elements. Viruses can exploit the cytoskeleton to promote different steps of their life cycle by hijacking distinct motor proteins. This review describes a few examples of the mechanisms that some human viruses have developed to subvert specific dyneins, kinesins and myosins.
Human adenoviruses (HAdV) are ubiquitous human pathogens that cause a significant burden of respiratory, ocular, and gastrointestinal illnesses. Although HAdV infections are generally self-limiting, pediatric and immunocompromised individuals are at particular risk for developing severe disease. Currently, no approved antiviral therapies specific to HAdV exist. Recent outbreaks underscore the need for effective antiviral agents to treat life-threatening infections. In this review we will focus on recent developments in search of potential therapeutic agents for controlling HAdV infections, with a focus on those targeting post-entry stages of the virus replicative cycle.
Gene therapy with human adenovirus type 5 [Ad5] has been extensively explored for the treatment of diseases resistant to traditional therapies. Following intravenous administration, Ad is rapidly cleared from systemic blood circulation with a half life of 2 minutes, and more than 99 % of the injected dose is sequestered in the liver. The resulting innate and adaptive immune responses dramatically affect the kinetics and toxicity profile of the vector. These issues currently restrict the use of Ad-based vectors, particularly for clinical gene therapy protocols that involve systemic administration. We propose that such limitations can be improved by engineering artificial lipid envelopes around Ad. We previously designed a variety of artificial lipid bilayer envelopes around the viral capsid. Zwitterionic and cationic lipid formulations can efficiently envelop Ad, however this resulted in a significant reduction of gene expression in vitro due to the inadequate escape of the enveloped virus from the endosomal compartment. In this thesis, pH-sensitive lipid-envelopes to enhance the virus release from the endosome following endocytosis were explored. Also, different viral envelopment methodologies (sonication and extrusion) were compared in terms of percentage of virion envelopment and efficiency of gene expression. The artificially enveloped Ad were characterised physicochemically by dot blots, dynamic light scattering and atomic force microscopy. Biologically, the gene expression of enveloped Ad in different pH-sensitive enveloped Ad was studied in vitro and in vivo. The critical role of blood components during systemic administration of Ad was investigated by looking at the interaction of enveloped Ad in cationic, non-pH-sensitive (DOTAP:Chol) or anionic, pH-sensitive [DOPE:CHEMS) lipid bilayers with several different blood components. When Ad was enveloped by cationic bilayers, significantly high levels of viral uptake in HepG2 cells were achieved, independent of any blood coagulation factor, whereas, the levels of cellular uptake and gene expression were similar to naked Ad vectors when an anionic lipid envelope was used. in vitro experiments also showed that artificial envelopment of Ad completely altered the affinity towards both human and murine red blood cells. After intravenous administration into mice, real-time PCR and transgene expression studies indicated that cationic lipid envelopes significantly reduced hepatocyte transduction compared to anionic envelopes. ALT/AST serum levels and liver histology showed that envelopment also improved hepatotoxicity profiles compared to naked Ad. Furthermore, envelopment in DOTAP:Chol lipid bilayers significantly increased lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. These results suggest that artificial envelopes for Ad significantly alter the interactions with blood components and divert viral particles from their natural liver tropism resulting in reduced hepatotoxicity. Finally, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within lipid bilayers. Such a dual-activity vector can possibly offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, ribo-green assays and dot blots showed that siRNA and Ad can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors. In summary, the studies in this thesis contribute to greater understanding of the mechanisms, obstacles and opportunities offered by artificial lipid envelopment of Ad and how these affect the biological activity of these promissing gene therapy vectors in vitro and in vivo.
Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. The human AdV (HAdV) species B and C, such as HAdV-C2, C5 and B14, cause respiratory disease, and constitute a health threat for immuno-compromised individuals. HAdV-Cs are well known for lysing cells, owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening frame-work and identified an inhibitor of HAdV-C2 multi-round infection, Nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of the human immuno-deficiency virus (HIV) aspartyl protease, and used to treat acquired immuno-deficiency syndrome (AIDS). It is not effective against single round HAdV infections. Here, we show that Nelfinavir inhibits the lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based trans-mission of cell-free viral particles from an infected cell to neighbouring uninfected cells. HAdV lacking ADP was insensitive to Nelfinavir, but gave rise to comet-shaped foci indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and B14p1 lacking ADP were highly sensitive to Nelfinavir, although HAdV-A31, B3, B7, B11, B16, B21, D8, D30 or D37 were less sensitive. Conspicuously, Nelfinavir unco-vered slow-growing round-shaped HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of non-lytic cell-to-cell transmission. Our study demonstrates the repurposing potential of Nelfinavir with post-exposure efficacy against different HAdVs, and describes an alternative non-lytic cell-to-cell transmission mode of HAdV.
Following receptor‐mediated uptake into endocytic vesicles and subsequent escape, adenovirus particles are transported along microtubules. The microtubule motor proteins dynein and one or more kinesins are involved in this behaviour. Dynein is implicated in adenovirus transport toward the nucleus. The kinesin Kif5B has now been found to move the adenovirus(AdV) toward microtubule plus ends, though a kinesin role in adenovirus‐induced nuclear pore disruption has also been reported. In undifferentiated cells, dynein‐mediated transport predominates early in infection, but motility becomes bidirectional with time. The latter behaviour can be modelled as a novel assisted diffusion mechanism, which may allow virus particles to explore the cytoplasm more efficiently. Cytoplasmic dynein and Kif5B have both been found to bind AdV through direct interactions with the capsid proteins Hexon, and Penton Base, respectively. We review here the roles of the microtubule motor proteins in AdV infection, the relationship between motor protein recruitment to pathogenic vs. physiological cargoes, the evolutionary origins of microtubule‐mediated AdV transport, and a role for the motor proteins in a novel host‐defense mechanism.
Since 2015, Fowl adenovirus serotype 4 (FAdV-4) infection has caused serious economic losses to the poultry industry worldwide. We isolated and identified the FAdV-4 strain NP, from infected chickens on a layer farm, using chicken embryo allantoic cavity inoculation, electron microscopy, viral genome sequencing, and regression analysis. To explore the pathogenesis of FAdV-4 infection, we conducted transcriptome sequencing analysis of the liver in chickens infected with FAdV-4, using the Illumina® HiSeq 2000 system. Two days after infection with the FAdV-4 NP strain, 13,576 differentially expressed genes (DEGs) were screened in the liver, among which, 7,480 were up-regulated and 6,096 were down-regulated. Gene ontology (GO) analysis indicated that these genes were involved in 52 biological functions. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that those DEGs were involved in 33 pathways. We then focused on the KEGG pathway of phagosome and found that mRNA levels of the 25 DEGs in that pathway were up-regulated, and seven DEGs were down-regulated. Real-time quantitative polymerase chain reaction (qPCR) confirmed the accuracy and reliability of these findings. Moreover, 24 h after LMH cells were infected with FAdV-4, the mRNA levels of F-actin, Rab7, TUBA, and DVnein were significantly increased. These four genes were all subsequently silenced by RNA interference, and viral replication of FAdV-4 was then significantly down-regulated. These findings demonstrate the isolation and identification of the FAdV-4 NP strain, and the DEGs in KEGG pathway of phagosome were utilized by FAdV-4 to benefit its infection.
Incoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on well‐coordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keeps uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.
More than 80 different adenovirus (AdV) types infect humans through the respiratory, ocular, or gastrointestinal tracts. They cause acute clinical manifestations or persist under humoral and cell-based immunity. Immuno-suppressed individuals are at risk of death from an AdV infection. Concepts about cell entry of AdV build on strong foundations from molecular and cellular biology—and increasingly physical virology. Here, we discuss how virions enter and deliver their genome into the nucleus of epithelial cells. This process breaks open the virion at distinct sites because the particle has nonisometric mechanical strength and reacts to specific host factors along the entry pathway. We describe how macrophages and dendritic cells resist AdV infection yet enhance productive entry into polarized epithelial cells. A deep understanding of the viral mechanisms and cell biological and biophysical principles will continue to unravel how epithelial and antigen-presenting cells respond to AdVs and control inflammation and persistence in pathology and therapy.
Expected final online publication date for the Annual Review of Virology Volume 6 is September 30, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Viruses are obligatory parasites that take advantage of intracellular niches to replicate. During infection, their genomes are carried in capsids across the membranes of host cells to sites of virion production by exploiting cellular behaviour and resources to guide and achieve all aspects of delivery and the downstream virus manufacturing process. Successful entry hinges on execution of a precisely tuned viral uncoating program where incoming capsids disassemble in consecutive steps to ensure that genomes are released at the right time, and in the right place for replication to occur. Each step of disassembly is cell-assisted, involving individual pathways that transmit signals to regulate discrete functions, but at the same time, these signalling pathways are organized into larger networks, which communicate back and forth in complex ways in response to the presence of virus. In this review, we consider the elegant strategy by which adenoviruses (AdVs) target and navigate cellular networks to initiate the production of progeny virions. There are many remarkable aspects about the AdV entry program; for example, the virus gains targeted control of a large well-defined local network neighbourhood by coupling several interacting processes (including endocytosis, autophagy and microtubule trafficking) around a collective reference state centred on the interactional topology and multifunctional nature of protein VI. Understanding the network targeting activity of protein VI, as well as other built-in mechanisms that allow AdV particles to be efficient at navigating the subsystems of the cell, can be used to improve viral vectors, but also has potential to be incorporated for use in entirely novel delivery systems.
La mucoviscidose est une maladie monogénique, caractérisée par des mutations survenant au niveau du gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Le clonage en 1989 du gène CFTR a permis d’envisager de traiter cette maladie par thérapie génique. Cela consiste à transférer à l’aide d’un vecteur, une version normale du gène CFTR dans les cellules atteintes des patients. En raison de la gravité des complications pulmonaires, c’est l’épithélium respiratoire qui constitue aujourd’hui le tissu cible pour le transfert de gènes. Le principe de la thérapie génique est évidemment très séduisant et un certain nombre d’essais cliniques ont d’ores et déjà été réalisés. La thérapie génique nécessite des outils de vectorisation efficaces et compatibles avec une utilisation répétée en clinique.Mon sujet de thèse a porté donc sur le développement, la biodistribution et l’optimisation de vecteurs synthétiques (lipides cationiques) pour le transfert de gènes dans l’épithélium respiratoire. Au cours de mes travaux, nous avons donc pu mettre au point des lipophosphoramidates KLN47 fluorescents utiles pour les études de biodistribution in vivo. Comparés au KLN47 non fluorescent, ces nouveaux composés présentent les mêmes propriétés physicochimiques, à savoir une taille relativement petite et un potentiel zêta positif. Sur lignées cellulaires, nous avons montré que les nouvelles formulations étaient aussi efficaces que le KLN47, et pas ou peu toxiques. Ensuite, sur modèle animal, les profils de biodistribution de lipoplexes pégylés et non-pégylés ont été comparés après injection systémique. Les profils de biodistribution des lipoplexes pégylés et non-pégylés étaient similaires, cependant, la pégylation des complexes a conduit à une circulation prolongée dans la circulation sanguine, alors que l’expression du transgène (luciférase) était équivalente dans les deux cas. De plus, l’activité luciférase était similaire à celle obtenue avec le KLN47 non fluorescent. Nous avons ainsi démontré que l’ajout des sondes lipidiques fluorescentes dans la solution liposomale du KLN47, ne modifie pas ses propriétés physicochimiques et transfectantes. L’ensemble des résultats montre que nous disposons d’outils prometteurs pour les études de biodistribution in vivo. D’autres molécules ont également été testées avec succès.
In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.
Gene therapy, the introduction of therapeutic genetic material to cells to ameliorate disease or injury, holds great potential for the treatment of numerous disorders. However, broad clinical application of gene therapy is currently hindered by the need for safer and more efficient delivery vectors. Vectors for gene delivery typically consist of a nucleic acid associated with a material, either natural (e.g., based on viruses) or synthetic (e.g., based on polymers or lipids). While viral vectors offer an attractive platform for gene delivery because of their efficient gene transfer capabilities, their clinical utility is often limited by the natural transduction patterns of viruses and, in many cases, their unfavorable interactions with the immune system. Various approaches have been pursued to address the challenges associated with therapeutic gene delivery, including efforts to tailor the properties of existing viral vectors through rational design and library-based selection (i.e., viral protein engineering), as well as strategies to assemble ‘artificial viruses’ from biological or synthetic building blocks. This chapter discusses current progress toward designing both viral and nonviral gene delivery vectors and describes future directions of the field, including the design of genetic cargo and the integration of gene vectors into biomaterial scaffolds.
Some viruses possess the remarkable ability to transport their genomes across nuclear pore complexes (NPCs) for replication inside the host cell’s intact nuclear compartment. Viral mechanisms for crossing the restrictive NPC passageway are highly complex and astonishingly diverse, requiring in each case stepwise interaction between incoming virus particles and components of the nuclear transport machinery. Exactly how a large viral genome loaded with accessory proteins is able to pass through the relatively narrow central channel of the NPC without causing catastrophic structural damage is not yet fully understood. It appears likely, however, that the overall structure of the NPC changes in response to the cargo. Translocation may result in nucleic acids being misdelivered to the cytoplasm. Here we consider in detail the diverse strategies that viruses have evolved to target and subvert NPCs during infection. For decades, this process has both captivated and confounded researchers in the fields of virology, cell biology, and structural biology.
The effectiveness of gene therapy is essentially governed by the ability of the vector to reach the relevant tissue, and once there, to facilitate the expression of appropriate quantities of the gene product. Over the course of evolution, viruses have developed highly specialized and successful methods to achieve those goals. For this reason, advances in gene therapy have mainly focused on the development of viral systems such as adenovirus (Ad), retrovirus, adeno associated virus, herpes virus, and others. This approach has been successful, as demonstrated by the recent proliferation of publications and human gene therapy trials involving various virus-based vectors. In particular, first generation replication-defective adenoviral vectors based on human serotypes from subgroup C (such as types 2 and 5) have been and continue to be very popular as vehicles for delivery and expression of foreign genes. However, each virus has evolved to optimize its own life cycle and consequently endows vectors based on it with properties and limitations that affect their suitability as choices for various gene therapy applications. These properties need to be understood and taken into account in order to design vectors with improved characteristics, such as Ad vectors with improved regulation of transgene transcription.
Targeted delivery of nucleic acid into disease sites of human body has been attempted for decades, but both viral and non-viral vectors are yet to meet our expectations. Safety concerns and low delivery efficiency are the main limitations of viral and non-viral vectors, respectively. The structure of viruses is both ordered and dynamic, and is believed to be key for effective transfection. Detailed understanding of the physical properties of viruses, their interaction with cellular components, and responses towards cellular environments leading to transfection would inspire the development of safe and effective non-viral vectors. To this goal, this review systematically summarizes distinctive features of viruses that are implied for efficient nucleic acid delivery but not yet fully explored in current non-viral vectors. The assembly and disassembly of viral structures, presentation of viral ligands, and the subcellular targeting of viruses are emphasized. Moreover, we describe the current development of cationic material-based viral mimicry and structural viral mimicry in these aspects. In light of the discrepancy, we identify future opportunities for rational design of viral mimics for the efficient delivery of DNA and RNA.
Adenoviruses contain an icosahedral coat and a nucleoprotein core of viral deoxyribonucleic acid. Mastadenoviruses infecting mammals are composed of several hundred different types. Human adenoviruses are the most widely used vectors in clinical applications, mainly for killing cancer cells and vaccinations. They are physically and genetically stable and are safe for use in humans. We discuss the state of the art in adenovirus entry, a key process for infection and immunity. Entry requires hundreds of host factors, but mechanisms are known for only some of them. It involves coating the virus with soluble serum or tissue factors, using receptors that initiate stepwise uncoating, signaling, endocytic uptake, and lysis of the endosomal membrane. Cytoplasmic transport and immunity, together with complete uncoating, determine nuclear import and transcriptional activation of the viral genome. We conclude with perspectives and a discussion of outstanding questions in the field.
Gene therapy, considered as treating genetically-caused diseases by transferring exogenous nucleic acids into specific cells of patients, has attracted great interests over the past few decades. Advances in molecular biology and biotechnology as well as the completion of the Human Genome Project have led to the recognition of numerous diseases-relating genes. It has been gradually and generally realized that development of safe, efficient and controllable gene-delivery vectors is now a bottleneck in clinical applications.
Treatment of cancer is limited by toxicity to normal tissue with standard approaches (chemotherapy, surgery and radiotherapy). The use of selective replicating viral vectors may enable the targeting of gene-modified viruses to malignant tissue without toxic effect. Studies of these vectors have demonstrated tumour-selective replication and minimal evidence of replication in normal tissue. The most advanced clinical results reported involve gene-modified adenoviral vectors. Several completed, histologically confirmed responses to local/regional injection have been induced, particularly in recurrent squamous cell carcinoma involving the head and neck region. Dose limiting toxicity above 10(13) viral particles per injection has been observed. Anti-tumour effect is demonstrable in animal models without evidence of significant toxicity when these vectors are used alone or in combination with chemotherapy, radiation therapy or as gene delivery vehicles. Preliminary clinical trials, particularly with E1B-deleted adenoviruses, report evidence of clinical activity in comparison with expected historical responses. Enhancement in replication selectivity to malignant tissue is also demonstrated preclinically and clinically with an E1B-deleted adenovirus utilising a prostate-specific antigen promoter. Other selective replicating viral vectors such as herpes simplex virus and vaccinia virus have also been explored clinically and suggest evidence of activity in patients with cancer. Modifications may one day enable more aggressive use of these new and exciting therapeutics as systemic gene delivery vehicles.
Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.
Replication-deficient adenoviral vectors (AdVec), which infect cycling and noncycling cells with high efficiency, low toxicity, and ease of delivery, provide ideal vehicles to study the expression of regulatory genes controlling different stages of hematopoiesis. To examine the infection efficiency of AdVec in hematopoietic precursor and progenitor cells, we used a replication-deficient adenovector expressing the humanized form of the cDNA for green fluorescent protein (AdGFP), permitting assessment of infection efficiency and kinetics of transgene expression in viable hematopoietic cells using flow cytometry and fluorescence microscopy. Flow-cytometric analysis of ex vivo expanded hematopoietic precursor cells infected with a multiplicity of infection (MOI) of 100 of AdGFP show that 78% of megakaryocytic (CD41a+ and CD42b+) cells, 82% of dendritic (CD1a+) cells, 41% of RBC precursors (glycophorin A+), and 32% of monocytic (CD14(+)) cells expressed GFP. Nineteen percent +/- 1% of freshly isolated CD34(+) cells from peripheral blood leukapheresis products infected under the same conditions expressed GFP. Morphologic evaluation of ex vivo expanded, AdGFP-infected CD34(+) cells showed normal maturation. The functional capacity of AdGFP-infected CD34(+) cells was analyzed by quantifying clonogeneic efficiency and proliferative capacity. Infection of CD34(+) progenitor cells with MOIs of 1 to 100 did not impair clonogeneic efficiency of CD34(+ )cells. However, MOI greater than 100 resulted in a significant inhibition of colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM) formation. In sequential dilution expansion over 3 weeks (Delta assay), the cytokine-driven proliferative potential of CD34(+) cells was not impaired following exposure to AdGFP at MOIs of 1 to 1,000. The GFP+ population expanded 10- to 15-fold at high MOIs (500 to 1,000), indicating multiple copies of the transgene in the initially infected CD34(+) cells, which were expressed in subsequent progenies. These data show that AdVec deliver transgenes with high efficiency and low toxicity to hematopoietic progenitor and precursor cells. Introduction of marker genes such as GFP into hematopoietic cells by AdVec will provide a valuable system for study of development, homing, and trafficking of hematopoietic precursor and progenitor cells in vitro and in vivo. Furthermore, these results provide insights into the design of gene therapy strategies for treatment of hematologic disorders by AdVec.
The cell receptors that facilitate adenovirus internalization into cells have been identified; however, the infectious pathway of virus entry has not been established. Adenovirus entry and infection were examined in HeLa cells lacking or overexpressing mutant dynamin, a protein that specifically regulates clathrin-mediated endocytosis. Expression of mutant dynamin significantly reduced adenovirus internalization and gene delivery, indicating a functional requirement for this molecule. These findings are consistent with virus entry via the clathrin-coated pit pathway.
Utilizing negative-stain electron microscopy in which similar concentrations of reovirus types 1 and 3 are incubated with a carbon support film containing chick brain, rabbit brain, or HeLa cell microtubules, 81% of the type 1 and 56% of type 3 exhibited an association with the apparent "edge" of the microtubule. This implies that there is a high level of specific affinity for type 1 but not for type 3 to microtubules, since it has previously been determined that only 50% of randomly associated particles would be associated with the edge. The high edge binding of reovirus type 1 is virtually independent of the origin of microtubule, or of whether microtubules or virus has been initially adhered to the support film. On the other hand, reovirus type 1-specific antiserum reduced the edge binding or reovirus type 1 to 45%, whereas type 3 specific antiserum caused no less (within the variability of the assay) of the edge binding of reovirus type 1 to microtubules (76% edge bound). High edge binding of reovirus type 1 to microtubules is correlated with the presence of type 1 or sigma 1 polypeptide. This minor outer capsid polypeptide is encoded in the S1 double-stranded RNA segment and is the viral hemagglutinin and neutralization antigen. Recombinant reovirus clones containing the S1 double-stranded RNA segment of type 1 (80 and 802) show about 85% edge binding, as compared to a value of 42% for clones and the S1 gene of type 3 (204. Electron microscopy of purified reovirus types 1 and 3 by negative staining reveals that type 1 and 802 capsomers are distinctly visualized, whereas those of type 3 and 204 appear diffuse. Thus, the greater in vitro binding of type 1 to microtubules may reflect an increased accessibility of certain of its outer capsomers, and thereby, sigma 1 polypeptides to microtubules. Examination of its outer sections of reovirus type 1- and 3-infected cells at 24 to 48 h postinfection at 31 degrees C showed that about eight times as many viral factoris in type 1-infected cells exhibited an extensive association of virus particles with microtubules, as compared to viral factories of type 3-infected cells. Thus, both in vivo and in vitro there appears to be a greater specificity for the association of reovirus type 1 particles with microtubules, as compared to reovirus type 3 particles.
A specific in vitro association between adenovirus and pruified rat brain microtubules has been previously demonstrated (R. B. Luftig and R. R. Weihing, 1975). When examined by negative-staining electron microscopy, approximately 90% of the virus associated with microtubules was edge bound, i.e., associated within +/-4 nm of the microtubule edge. Similar results are now found for the association of adenovirus with purified chick brain microtubules. When the content of the high-molecular-weight proteins (MAPs) normally present as projections on the surface of microtubules is depleted by fractionation of cold-depolymerized microtubules on agarose A-15M columns or by brief treatment of polymerized microtubules with trypsin, the percentage of edge-bound microtubule-associated viruses is reduced to a level close to that found for particles such as reovirus, coliphage f2, or polystyrene latex spheres, which randomly associate with microtubules (54 to 64% for column-fractionated microtubules; 45 to 68% for trypsin-treated microtubules). Counts of adenovirus particles specifically bound to microtubules, corrected for variations in microtubule and virus concentrations, gave values 2.5 to 3.5 times higher for unfractionated microtubules than for microtubule-associated protein-depleted microtubules. These results are consistent with the suggestion that the specific association between adenovirus and microtubules is mediated by microtubule-associated proteins.
The effect of colchicine, Nocodazole, and dibucaine on the assembly of Semliki Forest virus was investigated. Colchicine, Nocodazole, and dibucaine reduced the production of extracellular virus by 75 to 90%. Lumicolchicine had no effect on virus growth. Other control experiments showed no effect by these drugs on the incorporation of [3H]leucine into material precipitated by trichloroacetic acid. Colchicine (100 micron) disrupted the microtubles of the baby hamster kidney cells (BHK-21), whereas dibucaine did not alter microtubule polymerization. The stage of virus assembly inhibited by colchicine and dibucaine was studied by experiments with [3H]-leucine or [35S]methionine. At various times after addition of one of these drugs, the incorporation of the labeled precursors into viral proteins associated with fractions enriched for endoplasmic reticulum or plasma membrane from the cell was evaluated. The results clearly show that the envelope and nucleocapsid proteins of the virus move to the plasma membrane of the cell where they accumulate. The studies strongly suggest that the cytoskeletal system is involved in the final stages of morphogenesis of Semliki Forest virus from the plasma membrane.
We have found by negative staining electron microscopy that when similar concentrations of adenovirus and reovirus (viruses of about the same diameter, 75 to 80 nm, and density, 1.34 to 1.36 g/cm3) were incubated with a carbon support film containing microtubules, 72% of adenovirus on the grid, but only 32% (equivalent to random association) of reovirus, were associated with microtubules. Similar concentrations of both larger and smaller particles, such as polystyrene latex spheres and coliphage f2, also exhibited a low degree of interaction, viz., 17 to 37%, with microtubules. Moreover, 90% of microtubule-associated adenovirus binds to within +/- 4 nm of the edge of microtubules, but lower fractions (again equivalent to a random association) of the other particles bind to the edge of the microtubules. The mechanism behind this phenomenon, which we denote as "edge binding," is presently obscure; however, it provides us with a second, albeit empirical, method to distinguish between the microtubular association of adenovirus and other particles. We found that edge binding of adenovirus also occurred when adenovirus was initially placed on the carbon support film and then incubated with microtubules and when adenovirus and microtubules were mixed prior to placement on the support. In contrast, reovirus or the other particles prepared by similar techniques exhibited a random amount of edge binding. The binding of adenovirus appears to involve the hexon capsomers of the virion since (i) high resolution electron micrographs showed that the edge of the virus was in contact with the edge of the microtubules, and (ii) adenovirions briefly treated with formamide to remove pentons and fibers bind as efficiently as intact virions. Core structures, which were obtained by further formamide degradation of the virion, do not associate with microtubules. These observations support the hypothesis of Dales and Chardonnet (1973) that the transport of adenovirions within infected cells is mediated by interaction with microtubules.
One of our monoclonal antibodies against the heavy chain of bovine kinesin (H2) also recognized the heavy chain of squid kinesin. The immunofluorescence pattern of H2 in axoplasm was similar to that seen in mammalian cells with antibodies specific for kinesin light and heavy chains, indicating that squid kinesin is also concentrated on membrane-bounded organelles. Although kinesin is assumed to be a motor for translocation of membrane-bounded organelles in fast axonal transport, direct evidence has been lacking. Perfusion of axoplasm with purified H2 at 0.1-0.4 mg/ml resulted in a profound inhibition of both the rates and number of organelles moving in anterograde and retrograde directions in the interior of the axoplasm, and comparable inhibition was noted in bidirectional movement along individual microtubules at the periphery. Maximal inhibition developed over 30-60 min. Perfusion with higher concentrations of H2 (greater than 1 mg of IgG per ml) were less effective, whereas perfusion with 0.04 mg of H2 per ml resulted in minimal inhibition. Movement of membrane-bounded organelles after perfusion with comparable levels of irrelevant mouse IgG (0.04 to greater than 1 mg/ml) were not distinguishable from perfusion with buffer controls. Inhibition of fast axonal transport by an antibody specific for kinesin provides direct evidence that kinesin is involved in the translocation of membrane-bounded organelles in axons. Moreover, the inhibition of bidirectional axonal transport by H2 raises the possibility that kinesin may play some role in both anterograde and retrograde axonal transport.
Intermediate filaments in most types of cultured cells coalign with microtubules. Depolymerization of microtubules results in collapse of vimentin and desmin intermediate filaments to the nucleus where they form a perinuclear cap. Collapse can also be induced by microinjection of antibodies against intermediate filament or microtubule proteins. Thus, two filament systems interact with each other. But the molecules mediating this interaction are unknown. One of the candidates for this role is a microtubule motor kinesin. Recent data showed that kinesin is involved in the plus end-directed movement of the membranous organelles along microtubules such as radial extension of lysosomes in macrophages and centrifugal movement of pigment in melanophores. Here we report that injection of the anti-kinesin antibody into human fibroblasts results in the redistribution of intermediate filaments to a tight perinuclear aggregate but had no effect on the distribution of microtubules. Thus, kinesin is involved not only in organelle movement but also in interaction of the two major cytoskeletal systems, intermediate filaments and microtubules.
Internalization of the infectious fraction of human adenovirus type 2 into HeLa cells was followed by a quantitative internalization assay. Treatments known to selectively block receptor-mediated endocytosis reduced the internalization of infectious virus to an extent close to the reduction of endocytosis of transferrin. This suggests that one of the first steps in the infectious cycle of adenovirus type 2 is internalization by the coated-pit and -vesicle pathway.
We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 A, but may be as small as 200 A in some cytoplasmic domains.
Herpes simplex virus type 1 and a fluorescein-labelled lectin (wheat germ agglutinin) were selectively transported to nerve cell bodies located in the inner compartment of a two-chamber tissue culture system after the application of virus or lectin to the neuritic processes in the outer culture compartment. Taxol, which stabilizes and alters intracellular arrangements of microtubules, and nocodazole, which disrupts microtubules, both inhibited this retrograde axonal transport of viral particles and lectin. The transport was also inhibited by erythro-9-3-(2-hydroxynonyl)adenine (EHNA), which blocks ATPases. However, EHNA was also an effective inhibitor of infection with the virus in non-neuronal cells (GMK AH-1). The nature of the action(s) of EHNA on neuritic transport of the virus is therefore less clear.
Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport.
Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.
Cultured human fibroblasts showed a typical fibrillar organization of microtubules in immunofluorescence, including the vimentin type of intermediate filament as well as actin-containing microfilaments. During infection with herpes simplex virus type 1 (HSV-1), the vimentin organization was maintained whereas actin, myosin and tubulin showed a progressive association with the viral glycoproteins within juxtanuclear structures. These structures could also be revealed with fluorochrome-coupled wheat germ agglutinin. Disruption of the microtubules by demecolcine treatment or their stabilization by taxol treatment did not prevent the aggregation of viral proteins in the cytoplasm. Taxol stabilization of the microtubules allowed the juxtanuclear accumulation of the glycoproteins in HSV-infected cells whereas treatment with demecolcine led to an accumulation of the glycoproteins either in small vesicles in the cytoplasm or in the focal adhesion areas of the cells. Production of infectious intracellular virus particles was reduced in cells treated with demecolcine or with taxol before and during infection. The results of this study indicate that the normal intracellular transport and distribution of the HSV glycoproteins and the formation of infectious virus are dependent on the presence of intact microtubules.
We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.
We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.
Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 A in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 A, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.
The established positive cooperativity of adenovirus 2 binding to HeLa cells revealed a strong temperature dependence. The degree of cooperativity, quantified by means of Hill coefficients, progressively increased from 10 degrees C to reach a maximum level, which was maintained between 20 and 37 degrees C. On the other hand, negative cooperativity of virion attachment was apparent at 3.0 degrees C and on glutaraldehyde-stabilized cells. The corresponding monovalent ligand of the system, the fiber antigen, demonstrated only weak-positive cooperativity of the binding at 37.0 degrees C, which was absent at 3.0 degrees C. Dithiothreitol and dansylcadaverine, reagents inhibiting clustering of ligand-receptor complexes in the plasma membrane, markedly reduced the degree of positive cooperative binding at 37.0 degrees C. Evidently, the positive cooperative binding of adenovirus to HeLa cells at 37.0 degrees C is a consequence of both the multivalency of virus attachment proteins, i.e., fibers, on the virion and of the capacity of the receptor sites to migrate in the plane of the plasma membrane, forming local aggregates of virus-receptor site complexes.
Adenovirus uncoating is a stepwise process which culminates in the release of the viral DNA into the nucleus through the nuclear pore complexes and dissociation of the capsid. Using quantitative biochemical, immunochemical and morphological methods, we demonstrate that inhibitors of the cystine protease, L3/p23, located inside the capsid block the degradation of the capsid‐stabilizing protein VI, and prevent virus uncoating at the nuclear membrane. There was no effect on virus internalization, fiber shedding and virus binding to the nuclear envelope. The viral enzyme (dormant in the extracellular virus) was activated by two separate signals, neither of which was sufficient alone; virus interaction with the integrin receptor (inhibited with RGD peptides) and re‐entry of the virus particle into a reducing environment in the endosome or the cytosol. Incorrectly assembled mutant viruses that lack the functional protease (ts1) failed at releasing fibers and penetrating into the cytosol. The results indicated that L3/p23 is needed not only to assemble an entry‐competent virus but also to disassemble the incoming virus.
Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies, However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible, This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance, The methods for evaluation of the concentration of Functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells, Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption, volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability, These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.
Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin-dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus.
We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 A, but may be as small as 200 A in some cytoplasmic domains.
Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.
We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.
The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)--fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.
Combined biochemical and electron microscopic procedures were employed to follow the early interaction of adenovirus 5 with HeLa cells. Infection was started by first adsorbing the purified inoculum at low temperature, then initiating penetration by elevating the temperature. Within a few minutes attached particles were incorporated by viropexis, and became lodged in the vicinity of the nucleus. Initially the perinuclear particles possessing dense centers lay free in the cytoplasmic matrix, characteristically near nuclear pores. Later the dense perinuclear particles decreased in number and were replaced by capsid shells with lucent centers. This reconstructed morphological sequence was correlated with radiochemical data primarily on virus carrying 32P-label in the parental genomes. Analysis by cell fractionation and sucrose density gradients revealed that within 2 hours after warming most of the label had been transferred out of the cytoplasmic granule fraction and some became associated with nuclei. The labeled material could be extracted from purified nuclei and shown to possess the approximate density of adenovirus 5 DNA. Therefore, type 5 virus can penetrate rapidly and efficiently to the nuclear border where final uncoating and some transfer of the genome most probably commences in less than 1 hour.
The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre-Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex.
The pathogenic agent, adenovirus (Ad), has taken on a new role as a vector for gene transfer in both laboratory and clinical settings. To help understand the intracellular pathways and fate of Ad gene transfer vectors, we covalently conjugated fluorophores to E1-, E3- Ad vectors and used quantitative fluorescence microscopy to assess essential steps of Ad vector gene transfer to the A549 human epithelial lung cell line including binding, internalization, escape from endosomes, translocation to the nucleus, dissociation of capsids and gene expression. The data demonstrate that Ad internalizes with a t1/2 2.5 min, breaks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 microm/sec, and translocates to the nucleus with >80% of internalized fluorophore demonstrating nuclear localization within 60 min of infection. Interestingly, 24 hr after infection, half of the initially internalized fluorescence was detected but lacked nuclear localization, suggesting that the capsid is released from the nucleus and is likely degraded. Fluorescent labeling of virions provides a novel quantitative, morphological strategy to characterize the interaction of gene transfer vectors with the intracellular environment.
The uncharged, colorless molecule fluorescein diacetate diffuses into Ehrlich ascites tumor cells at neutral pH, where intracellular esterases release the chromophore fluorescein. The negatively charged dye is retained by the cell, permitting the intracellular pH to be estimated from the shape of the pH-dependent absorption spectrum. The diacetate derivative of 6-carboxyfluorescein may be used similarly and has the additional advantage of a slower rate of leakage out of the cell but requires incubation at pH 6.2 to facilitate initial entry into the cell. After removal of external dye by centrifugation, 80-92% of the remaining dye is unresponsive to external pH changes. Calibration of the intracellular fluorescein spectra is obtained by equilibration of the internal and external pH with nigericin in K+ buffers. Results of intracellular pH measurements by this method are in good agreement with those obtained by measuring the distribution ratio of the weak acid 5,5-dimethyl[2-14C]oxazolidine-2,4-dione, under a variety of metabolic conditions. Besides the accurate estimation of intracellular pH, the method permits the kinetics of intracellular pH changes as small as 0.01 to be followed. Intracellular fluorescein reports pH changes occurring in both the cytoplasmic and the mitochondrial compartments, whereas 6-carboxyfluorescein reports only the cytoplasmic compartment. At equivalent concentrations, nigericin is more effective than valinomycin plus the protonophore 1799 in dissipating plasmalemma pH gradients. Either is effective at lower concentrations in dissipating mitochondrial pH gradients. Addition of glucose to Ehrlich ascites cells results in a transient acidification of the cytoplasm in close correspondence to the intracellular lactate levels. The transient acidification can be explained by the initial rapid rate of glycolysis exceeding the rate of lactate export.
Previous studies revealed that, during adenovirus penetration toward the nucleus, there is no obvious alteration in the appearance of inoculum particles until the uncoating phase has occurred in the vicinity of the nuclear pore complex. Early interactions of adenovirus 5 with HeLa cells were again examined on the premise that subtle, yet morphologically undetectable, changes might take place and can be associated with the removal of a specific capaid component(s). Data from autoradiography show that DNA from inoculum virions labeled with [3H]thymidine enters the nucleus rapidly and efficiently but the bulk of the [3H]leucine-labeled protein remains behind in the cytoplasm. Following isolation of 35S-labeled particles from cell-virus complexes by means of sucrose gradients, analysis of virion polypeptides was conducted by polyacrylamide gel electrophoresis (PAGE). With the possible exception of minor changes in stoichiometric quantities of the fiber or penton + fiber component, the patterns of polypeptides from purified virus or from virus attached to the plasma membrane or isolated from the cytoplasm were very similar. This finding is also valid for the polypeptide spectrum obtained after analysis of intracytoplasmic virions isolated as complexes with microtubule paracrystals. These observations focus attention on the possible function of individual capaid components during internalization and vectorial transfer of the inoculum prior to uncoating.
Microtubules are closely associated with tubules containing virus particles in leaf cells infected with bean pod mottle virus or cowpea mosaic virus. The similar orientation and apparent continuity suggest that cores of tubules with virus particles are actually microtubules. It is suggested that microtubules may be involved in the intra- and intercellular movement of viral components and in the assembly of virus particles as well as being a factor in the reaction of a plant to virus infection.
Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.
The sulfhydryl groups of tubulin are highly reactive entities. The reactivity of the sulfhydryl groups is sensitive to the presence of tubulin ligands, making these groups excellent probes for the interaction of tubulin with ligands. When tubulin is reacted with N,N'-ethylenebis-(iodoacetamide), two intrachain cross-links form in the beta subunit. Formation of one of these cross-links is completely blocked by colchicine, podophyllotoxin, and nocodazole; formation of the other is blocked completely by maytansine, phomopsin A and GTP and partly by Vinca alkaloids. Different ligands also differ in their effect on the rate of alkylation of tubulin with iodo[14C]acetamide, with vinblastine and phomopsin A being strong inhibitors and maytansine having very little effect. Oxidation of certain key sulfhydryl groups can inhibit microtubule assembly. One of these sulfhydryl groups appears to be cys239, but there are others not yet identified. Sulfhydryl-oxidizing agents also interfere with microtubule-mediated processes in vivo, raising the question of the existence of a physiological regulator of microtubule assembly. Potential physiological regulators have been examined to see if they can control microtubule assembly in vitro at their physiological concentrations. Of the ones that have been examined, thioredoxin and thioredoxin reductase are much better candidates for being physiological regulators than are either cystamine or glutathione.
The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin
(alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus
major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory
tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human
alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration
of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was
synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.
Estramustine phosphate, an estradiol-mustard conjugate, was shown to reversibly inhibit a stage during the first hour of productive adenovirus 2 infection of HeLa cells. This drug, employed in the therapy of advanced prostatic cancer, specifically interacts with microtubule-associated proteins (MAPs) of the cytoskeleton. The results obtained under physiological conditions in vivo suggest a MAPs-interference with the microtubule-mediated vectorial migration of the virus inoculum to the nucleus. Virus attachment, uncoating kinetics and the appearance of established uncoating intermediates were not affected.
Host cell interactions of human adenovirus serotypes belonging to subgroups B (adenovirus type 3 [Ad3] and Ad7) and C (Ad2 and Ad5) were comparatively analyzed at three levels: (i) binding of virus particles with host cell receptors; (ii) cointernalization of macromolecules with adenovirions; and (iii) adenovirus-induced cytoskeletal alterations. The association constants with human cell receptors were found to be similar for Ad2 and Ad3 (8 x 10(9) to 9 x 10(9) M-1), and the number of receptor sites per cell ranged from 5,000 (Ad2) to 7,000 (Ad3). Affinity blottings, competition experiments, and immunofluorescence stainings suggested that the receptor sites for adenovirus were distinct for members of subgroups B and C. Adenovirions increased the permeability of cells to macromolecules. We showed that this global effect could be divided into two distinct events: (i) cointernalization of macromolecules and virions into endocytotic vesicles, a phenomenon that occurred in a serotype-independent way, and (ii) release of macromolecules into the cytoplasm upon adenovirus-induced lysis of endosomal membranes. The latter process was found to be type specific and to require unaltered and infectious virus particles of serotype 2 or 5. Perinuclear condensation of the vimentin filament network was observed at early stages of infection with Ad2 or Ad5 but not with Ad3, Ad7, and noninfectious particles of Ad2 or Ad5, obtained by heat inactivation of wild-type virions or with the H2 ts1 mutant. This phenomenon appeared to be a cytological marker for cytoplasmic transit of infectious virions within adenovirus-infected cells. It could be experimentally dissociated from vimentin proteolysis, which was found to be serotype dependent, occurring only with members of subgroup C, regardless of the infectivity of the input virus.
Identification of the ATPase involved in fast axonal transport of membranous organelles has proven difficult. Myosin and dynein, other ATPases known to be involved in cell motility, have properties that are inconsistent with the established properties of fast axonal transport, an essential component of which is readily solubilized in physiological buffer conditions rather than being stably associated with either membranous organelles or cytoskeletal elements. Adenylyl imidodiphosphate (AMP-PNP), a nonhydrolysable analogue of ATP, is a potent inhibitor of fast axonal transport that results in a stable interaction of membranous organelles with microtubules. Here we report the identification and partial characterization of an ATPase activity from brain whose binding to microtubules is stabilized by AMP-PNP. This ATPase activity seems to be associated with a polypeptide of relative molecular mass (Mr) 130,000 that is highly enriched in microtubule pellets after incubation with AMP-PNP and a soluble fraction from chick brain. This novel ATPase fraction has the predicted characteristics of the motor involved in fast axonal transport. Common features between the ATPase and fast axonal transport include interaction with the cytoskeleton in the presence of AMP-PNP, ready extractability, no Ca2+ dependence and inhibition by EDTA.
Single microtubules from squid axoplasm support bidirectional movement of organelles. We previously purified a microtubule translocator (kinesin) that moves latex beads in only one direction along microtubules. In this study, a polar array of microtubules assembled off of centrosomes in vitro was used to demonstrate that kinesin moves latex beads from the minus to the plus ends of microtubules, a direction that corresponds to anterograde transport in the axon. A crude solubilized fraction from squid axoplasm (S1a), however, generates bidirectional movement of beads along microtubules. Retrograde bead movement (1.4 micron/sec) is inhibited by N-ethylmaleimide and 20 microM vanadate while anterograde movement (0.6 micron/sec) is unaffected by these agents. Furthermore, a monoclonal antibody against kinesin, when coupled to Sepharose, removes the anterograde, but not the retrograde, bead translocator from S1a. These results indicate that there is a retrograde bead translocator which is pharmacologically and immunologically distinct from kinesin.
The role of cytoplasmic dynein in microtubule-based organelle transport was examined using a reconstituted assay developed from chick embryo fibroblasts. Factors present in a high-speed cytosol caused the movement of purified organelles on microtubules predominantly in the minus end direction. Inactivation of cytoplasmic dynein in the high-speed cytosol by vanadate-mediated UV photocleavage inhibited minus end-directed organelle motility by over 90%. Addition of purified cytoplasmic dynein to the inactive cytosol restored minus end-directed organelle motility, although purified cytoplasmic dynein by itself did not support organelle movement. We propose that cytoplasmic dynein is the motor for minus end-directed organelle movement, but that additional cytosolic factors are also required to produce organelle motility.
In this investigation, the early period of adenovirus type 2 (Ad2)-HeLa cell interaction was analyzed by electron microscopy and biochemical techniques. Events observed in this period ranged from the disappearance of virions from the cell surface to their subsequent association with the cell nucleus. Destabilization of the virions attached to the intact cell was necessary for virions to escape from intracellular vesicles. Strong temperature dependence and rapid escape from a vesicular compartment were shown in temporal kinetic experiments. These vesicles appeared to be acidic, since lysosomotropic agents partly inhibited the release of virions from vesicles. Studies of Ad2 binding to cells in buffers of different pH values suggested that adenovirus binds to cells by two different mechanisms. At low pH the binding was most probably mediated by the penton base and at neutral pH by the fiber protein. The number of receptor sites per cell was 25,000 and 6,000 at low and neutral pH, respectively. This study suggests that the low-pH affinity between the penton base and a vesicular membrane is important inside acid vesicles when Ad2 quickly enters the cytoplasm. However, a significant fraction of the virions was possibly internalized by a pathway not requiring a passage through such vesicles.
To understand the molecular basis of microtubule-associated motility during mitosis, the mechanochemical factors that generate the relevant motile force must be identified. Myosin, the ATPase that interacts with actin to produce the force for muscle contraction and other forms of cell motility, is believed to be involved in cytokinesis but not in mitosis. Dynein, the mechanochemical enzyme that drives microtubule sliding in eukaryotic cilia and flagella, has been identified in the cytoplasm of sea urchin eggs, but the evidence that it is involved in cytoplasmic microtubule-based motility (rather than serving as a precursor for embryonic cilia) is equivocal. Microtubule-associated ATPases have been prepared from other tissues, but their role in cytoplasmic motility is also unknown. Recent work on axoplasmic transport, however, has led to the identification of a novel mechanochemical protein called kinesin, which is thought to generate the force for moving vesicles along axonal microtubules. These results suggest that kinesin may also be a mechanochemical factor for non-axoplasmic forms of microtubule-based motility, such as mitosis. We describe here the identification and isolation of a kinesin-like protein from the cytoplasm of sea urchin eggs. We present evidence that this protein is localized in the mitotic spindle, and propose that it may be a mechanochemical factor for some form of motility associated with the mitotic spindle.
Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.
Intracytoplasmic type A particles known to be precursors to type B retroviruses in murine, hamster and marsupial cells are closely associated with microtubules and microtubule organizing centres. In this publication, the active participation of microtubules in the intracellular transport of the particles to the cell surface has been examined in NIH 3T3 cells infected with M432 virus using vincristine sulphate (VCR) as inhibitor of microtubule polymerization. The release of virus at different times after exposure to VCR was quantified by reverse transcriptase determinations of cell supernatants and by electron microscopic quantification of the number of virions at the cell surface using freeze-dried whole cell replicas. These studies indicate that VCR inhibits both microtubule polymerization and virus release, and thus suggest that intact cytoplasmic microtubules are necessary for intracellular transport and release of virus.
Morphological evidence was obtained indicating that transfer of cores from inoculum particles into nuclei of HeLa cells occurs at the nuclear pore complex (NP). When nuclei are released by means of detergent from cells sampled 30 min after initiating penetration, only particles with dense cores are attached to NP. In material taken at later times, empty attached capsids are evident indicating that inoculum virus becomes complexed firmly with the NP where transfer may occur.Quasi-intact nuclear envelopes were isolated by a new procedure from uninfected HeLa cells. They contain an Mg2+-requiring ATPase, which is optimally active at neutrality and has a Michaelis constant of 1.2 × 10−4M. Localization of the ATPase activity in isolated envelopes was carried out by means of electron microscopic cytochemistry. Presence of dense precipitates, presumed to represent the reaction product, was found in the vicinity of NP. A hypothesis currently favored by us postulates that an energetic process existing in the NP is involved with transfer of adenovirus cores and fulfills a more general role in nucleocytoplasmic movement of materials.
Only minutes elapse before adenovirus 5 (AV5) particles are transferred from the cell surface to the perinuclear site of uncoating. Morphological evidence suggesting that virions are moved vectorially along pathways provided by the microtubules is supported by experiments with vinblastine, which induces the formation of microtubular paracrystals (PC): intimate attachment of AV5 to PC was observed within intact cells or following isolation of virion-PC complexes and was inferred after mixing in vitro PC and AV5 suspensions. Together with data showing a delay in the initiation of the infectious cycle by vinblastine, these observations imply that when microtubules become concentrated within PC, normal transfer of the inoculum to the nucleus is, at least in part, affected. On the other hand, failure to interrupt appreciably the flow of AV5 to the nucleus by colchicine appears to be inconsistent with the above finding and may imply that intact microtubules are not involved in the vectorial movement. The nuclear pore complex (NP) was further identified as the specific ultimate site of intracellular AV binding since (a) colchicine elicited the formation of NP-like organelles in the cytoplasm to which inoculum virions are bound; (b) virions were isolated as part of a stable complex with NP on nuclei released from recently infected cells and, furthermore, the AV5-NP association survived isolation and purification of the nuclear envelopes. Application of p-hydroxymercuric benzoate at nontoxic concentrations suppressed both the uncoating and an ATPase activity of nuclear envelopes present at or near the NP. This result provides further circumstantial evidence in support of our previous hypothesis stating that AV uncoating at the NP is mediated by an ATPase.
The three antigens associated with adenovirus type 5 have been purified and investigated by electron microscopy and immunology. Antigen A is a round object 80 Å in diameter. Antigen B has a round head 80 Å in diameter attached to a tail 200 Å long and 20 Å wide with a 40 Å knob on the end. Antigen C is like antigen B without its head. Trypsin digests the head of antigen B and converts antigen B to antigen C.Antigen A forms the 240 capsomers† on the adenovirus surface that have 6 nearest neighbours. The other 12 capsomers on the vertices are antigen B and have 5 nearest neighbours. The tail on each antigen B extends outwards from the virus surface. Antigen C exists in the virion only as part of antigen B.