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Human adenovirus-host cell interactions: Comparative study with members of subgroups B and C
Journal of Virology
Abstract
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.
... HA, hemagglutinin; HRV, human rhinovirus ; KSH, K-acetate, sucrose, and Hepes. son et al., 1968 ) but is internalized through vitronectin-binding integrins by receptor-mediated endocytosis (FitzGerald et al., 1983; Defer et al., 1990; Wickham et al., 1993). Subsequently, penetration into the cytosol from the early endosome (Greber et al., 1993) is triggered by the acidic pH prevailing in this compartment (Pastan et al., 1986). ...
... Analysis by EM showed these cytoplasmic viral particles to be morphologically intact; however, recent biochemical and immunochemical data revealed the gradual dismantling of the virus during entry into the cytoplasm (Greber et al., 1993). Moreover, viral penetration results in the release of cointernalized proteins or DNA into the cytoplasm (FitzGerald et al., 1983; Pastan et al., 1986; Defer et al., 1990; Yoshimura et al., 1993 ). Adenovirus has also been shown to increase the permeability of the plasma membrane for small molecules when the cells are placed under mildly acidic conditions (Seth et al., 1984Seth et al., , 1985Seth et al., , 1987), and it can cause lysis of liposomes (Blumenthai et al., 1986) and the release of choline from isolated plasma membrane vesicles at low pH (Seth, 1994). ...
... Having demonstrated the applicability of this assay for the investigation of the effect of viral peptides on endosome integrity, we next determined the effect of adenovirus on endosomes. Adenoviruses of the subgroup C (AD2 and AD5) have been shown to disrupt the membrane of endocytic vesicles, thereby releasing cointernalized molecules such as proteins and nucleic acid into the cytoplasm (FitzGerald et al., 1983; Pastan et al., 1986; Defer et al., 1990; Curie1 et al., 1991; Yoshimura et al., 1993 ). Disruption of the membrane is strongly favored by the low pH of the endosome (Seth et al., 1984; Greber et al., 1993). ...
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.
... Studies of entry and intracellular trafficking of species B have been done mainly with Ad3 [292] and Ad7 [287,292,293] and to some extent using pseudotyped Ad-vectors expressing predominantly the species C capsid and species B fiber, or parts of the fiber, such as Ad5/Ad7f [294] and Ad5/Ad35fk [295]. Ad3 infection of malignant melanoma M21-L4 cells expressing v 3 and v 5 has been shown to be inhibited by a combination of antibodies directed at the v integrins, thus indicating that at least Ad3 of the species B adenoviruses depend on integrin co-receptors for internalization [259]. ...
... Studies of entry and intracellular trafficking of species B have been done mainly with Ad3 [292] and Ad7 [287,292,293] and to some extent using pseudotyped Ad-vectors expressing predominantly the species C capsid and species B fiber, or parts of the fiber, such as Ad5/Ad7f [294] and Ad5/Ad35fk [295]. Ad3 infection of malignant melanoma M21-L4 cells expressing v 3 and v 5 has been shown to be inhibited by a combination of antibodies directed at the v integrins, thus indicating that at least Ad3 of the species B adenoviruses depend on integrin co-receptors for internalization [259]. ...
... It was earlier reported that species B (Ad3 and Ad7) and species C (Ad2 and Ad5) used different internalization routes [292,293]. Ad5 is released early from endosomes at ~pH6.0, and Ad7 is released late from endosomes or endolysosomes at ~pH5.5 [287]. The Ad7 fiber seems to be the pH-dependent modulator of endosomal escape, since a pseudotyped Ad5 capsid expressing Ad7 fiber showed a similar endocytic transportation pattern as Ad7 [287,294]. ...
... Thus, Ad–cell binding in step (i) is mediated by the terminal sphere of the fiber (the fiber knob) protruding from the penton base capsomere and fiber receptors present at the cell surface (Bergelson et al., 1997; Hong et al., 1997; Tomko et al., 1997 ). The knob serotype is therefore a major determinant of the cell tropism (Defer et al., 1990; Krasnykh et al., 1996; Santis et al., 1999; Stevenson et al., 1995; Xia et al., 1995), even though it is probably not the only viral factor (Roelvink et al., 1998 ). At the second step (ii), the interaction of RGD and LDV triplet motifs in the penton base with the cell plasma membrane integrins is required to induce or trigger endocytosis. ...
... mechanisms of endosomal escape (v), transcytoplasmic movement (vi), and traverse of the nuclear pore channel (vii) still remain elusive. However, involvement of the penton base in endosomal escape has been assessed (Seth, 1994a; Seth et al., 1984; Yoshimura, 1985; Yoshimura et al., 1993 ), as has the participation of cytoskeletal elements in step vi ( Boulanger, 1985, 1987; Dales and Chardonnet, 1973; Defer et al., 1990; Weatherbee et al., 1977; Zhai et al., 1988; Zhang and Schneider, 1994 ). Furthermore, a functional nuclear localization signal has been identified in hexon, the major Ad capsid protein (Saphire et al., 1995), which could account for the binding of partially uncoated adenoviral DNA, still associated with hexons and core proteins (Greber et al., 1993), to the nuclear pore complex (NPC). ...
... The penton base activity was abolished after being heated to 56°C for 30 min, suggesting that it required native protein conformation. These results suggested that isolated penton base protein facilitated the internalization of macromolecules, as does the adenovirion itself (Defer et al., 1990; Karayan et al., 1997), but with a much lower efficiency. Considering that about 1% of the penton base input entered the cells, and that one infecting unit of Ad2 (1 PFU) corresponded to about 30–50 physical particles containing 12 penton capsomeres each, the level of endosomal release of RCA–Tf was 1000 to 2000 times more efficient with virions than with free penton base protein . ...
An Ad2 capsid component, the penton base, expressed as recombinant protein, was found to be capable of affecting the entire entry pathway of adenovirion in HeLa cells, i.e., cell attachment, endocytosis, vesicular escape, intracytoplasmic movement, and translocation through the nuclear pore complex. Data with pentamerization-defective mutants suggested that none of these successive steps depended upon penton base pentamer status, indicating that the peptide domains responsible for these functions were carried by the monomer. Observations performed with wild-type (WT) and an integrin-binding-site double-mutant (K288E340) suggested that the penton base could enter the cell via an alternative, RGD- and LDV-independent, pathway. Of three mutants that were found to be defective in nuclear addressing in insect cells, only one, W165H, was also altered in nuclear transport in HeLa cells. The other two, W119H and RRR547EQQ, showed a WT pattern of nuclear localization in HeLa cells, suggesting that the region including tryptophan-119 and the basic signal at position 547 did not act as a nuclear localization signal in the human cell context. The integrity of cellular structures and the cytoskeleton seemed to be required for the vectorial movement and nuclear import of WT penton base, as suggested by experiments using permeabilized HeLa cells, isolated nuclear membranes, and cytoskeleton-targeted drugs.
... Therefore, 7000 virus receptors per cell should be exposed. These results were in accordance with previous estimates from the KB, A549, and HeLa cells infected by Ad3 or Ad2 (Defer et al., 1990;Lonberg-Holm et al., 1976). ...
... Further studies confirm that adenovirus internalization was mediated by ␣ v (Mathias et al., 1994;Nemerow et al., 1994) and 2 integrins (Huang et al., 1996). Regarding the primary fiber attachment to the host cell, Defer et al. (1990) first suggested that Ad2 and Ad3 virions could bind to different receptors on KB and A549 cells, based on competition binding experiments using intact adenovirus particles and the virus overlay protein blotting assay. However, it was not possible to perform the reciprocal competition experiments, due to insufficient amounts of Ad2 and Ad3 fibers available. ...
... Svensson et al. (1981Svensson et al. ( , 1985 selected plasma membrane glycoproteins by affinity to wheat germ agglutinin and found that a 40-to 42-kDa glycoprotein from HeLa cells interacted with Ad2 virions. Defer et al. (1990) reported that numerous membrane proteins with various molecular weights bind Ad3 virions and that Ad2 and Ad3 were found to bind to different proteins. Di Guilmi et al. (1995) demonstrated that two HeLa membrane proteins, with molecular masses of 130 and 100 kDa, were specifically bound by Ad3 virions using a virus overlay protein blotting assay. ...
The host–cell interactions of the genome types Ad11p and Ad11a of human adenovirus serotype 11, displaying kidney or respiratory tropism, were compared using FACS analysis. Kinetic experiments indicated that the virus binding started immediately and reached a plateau after 30 min. The binding of biotinylated virions to seven continuous cell lines: A549, A498, J82, HeLa, CHO, MDCK, and human diploid fibroblasts (HEDF), was quantitated by FACS analysis. The binding capacities of the two viruses to all human cell lines but A549 cells appeared to differ. Ad11p virions manifested high affinities, whereas Ad11a virions presented low affinities. Neither of the two viruses bound to CHO or MDCK cells. Reciprocal competition experiments showed that the Ad11a virions could be weakly blocked by the Ad11p virions, whereas the Ad11p virions could not be competed at all by the Ad11a virions. The binding of the Ad11p virions to cells could be blocked by the rfiber antiserum of Ad11p, but not by the corresponding antiserum against Ad11a or Ad35p. A comparison of the cytopathogenicity of the seven cell lines infected by Ad11p and Ad11a demonstrated that the efficiency of the initial event of an adenovirus infection directly affects the outcome of the viral infection. The Ad11a in the A498, J82, HeLa, or HEDF cells that presented lower affinity and receptor concentration showed 100 times less infectivity than that in A549 cells displaying high affinity and receptor concentration. These results indicate that the cell susceptibility to Ad11p and Ad11a infection strongly depends on both the number of fiber receptors on the host cells and the receptor affinity for ligands on the fiber knob. Our findings also suggest that the receptors for Ad11p and Ad11a on the surface of different cell types may be different or on different sites.
... VIM rearrangement contributes to the formation of CSFV VRC. Previous reports have shown that VIM rearrangement induced the formation of cage-like structures for promotion of efficient viral RNA replication (29,30). To gain insight into the interaction between VIM and CSFV replication, cells infected with CSFV (MOI = 1) were fixed with 4% paraformaldehyde at 12, 24, and 36 hpi and stained with the indicated antibodies for confocal microscopy. ...
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF.
... Adenoviruses are DNA viruses that typically cause mild infections involving the upper or lower respiratory tract (Berk, 1991;Lynch and Kajon, 2016). Studies have shown that the adenoviral serotypes requiring endosome independent transcytoplasmic penetration routes have proteases that cleave vimentin intermediate filaments (Belin and Boulanger, 1987;Defer et al., 1990) (Figure 2). Although the function of cleaved vimentin is not described in these studies, it is possible that cleaved vimentin could transport the adenovirus directly to the perinuclear region due to collapse of vimentin intermediate filament network similar to HIV-1 infections (Thomas et al., 1996). ...
Vimentin intermediate filaments, a type III intermediate filament, are among the most widely studied IFs and are found abundantly in mesenchymal cells. Vimentin intermediate filaments localize primarily in the cytoplasm but can also be found on the cell surface and extracellular space. The cytoplasmic vimentin is well-recognized for its role in providing mechanical strength and regulating cell migration, adhesion, and division. The post-translationally modified forms of Vimentin intermediate filaments have several implications in host-pathogen interactions, cancers, and non-malignant lung diseases. This review will analyze the role of vimentin beyond just the epithelial to mesenchymal transition (EMT) marker highlighting its role as a regulator of host-pathogen interactions and signaling pathways for the pathophysiology of various lung diseases. In addition, we will also examine the clinically relevant anti-vimentin compounds and antibodies that could potentially interfere with the pathogenic role of Vimentin intermediate filaments in lung disease.
... Subgroup B serotypes accumulate in lysosomes, whereas subgroup C serotypes traffic rapidly to the nuclear envelope, revealing different kinetics of endosomal escape. Subgroup C serotypes' optimum pH for membrane lysis matches the one of early sorting endosomes, while subgroup B serotypes' optimum matches the pH of late endosomes or lysosomes [26]. Thus, HAdV intracellular trafficking is clearly determined by the initial interaction with the cellular receptor. ...
Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking; how they influence efficiency of adenovirus-based vectors; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors.
... If species C viruses (Ad5) are engineered to contain a fiber molecule from a species B virus (Ad16), the resulting particle (Ad5F16) then escapes from a lysosomal compartment (as shown by costaining with the lysosomal marker LAMP1) instead of the early endosome [351]. This delayed escape from lysosomes is a feature shared with all tested species B viruses [111,352,353]. Furthermore, the hybrid Ad5F16 as well as species B viruses elicit a stronger pro-inflammatory response than species C viruses [159,351]. ...
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.
... Vimentin has been shown to facilitate efficient viral RNA replication and viral protein synthesis by concentrating around the viral factories and form cage-like structures, termed vimentin cages. (Murti et al., 1988;Defer et al., 1990;Teo and Chu, 2014) (Fig. 2A) (Table 1). Confocal microscopy analysis showed that vimentin filaments and the Golgi concentrate in viral factories, where vimentin colocalizes with the core protein p39 of the vaccinia virus (VV) (Ferreira et al., 1994;Risco et al., 2002). ...
Epidemics caused by viral infections pose a significant global threat. Cytoskeletal vimentin is a major intermediate filament (IF) protein, and is involved in numerous functions, including cell signaling, epithelial–mesenchymal transition, intracellular organization and cell migration. Vimentin has important roles for the life cycle of particular viruses; it can act as a co-receptor to enable effective virus invasion and guide efficient transport of the virus to the replication site. Furthermore, vimentin has been shown to rearrange into cage-like structures that facilitate virus replication, and to recruit viral components to the location of assembly and egress. Surprisingly, vimentin can also inhibit virus entry or egress, as well as participate in host-cell defense. Although vimentin can facilitate viral infection, how this function is regulated is still poorly understood. In particular, information is lacking on its interaction sites, regulation of expression, post-translational modifications and cooperation with other host factors. This Review recapitulates the different functions of vimentin in the virus life cycle and discusses how they influence host-cell tropism, virulence of the pathogens and the consequent pathological outcomes. These insights into vimentin–virus interactions emphasize the importance of cytoskeletal functions in viral cell biology and their potential for the identification of novel antiviral targets.
... Virus internalisation then occurs by receptor-mediated endocytosis (Chardonnet 1970, Fitzgerald 1983. Furthermore, adenovirus particles stimulate the internalisation of macromolecules into host cells (Defer 1990). This last characteristic was suggested as a possible mechanism to increase ODN uptake. ...
This thesis reviews injurious events in the transplanted lung, describes the clinical correlates of these, and presents two novel therapeutic approaches. The mechanisms of injury in the transplanted lung have been detailed with special reference to allograft ischaemia, rejection, and infection. Clinical correlation of these injurious factors incorporated an extensive review of pulmonary function in a cohort of paediatric lung transplant recipients with cystic fibrosis. This study demonstrated an improvement in all dynamic lung function parameters at 12 months post transplant compared with pre transplant values (p0.001). Subsequently, there was a persistent decline in these values denoting the development of obliterative bronchiolitis. Measuring area under the curve of FEV1% graphs (FEV1% AUC) represented a unique method for assessing pulmonary function. This correlated negatively with graft ischaemic times (cold: p0.05, and total: p0.01) to 36 months post transplant, although not with the number of rejection or infection episodes in the first 12 months post transplant. Novel therapeutic approaches included the evaluation of a non-viral, synthetic peptide gene vector system. This incorporated the administration, into the airway, of a ligand-polylysine (polylysine-molossin) vector in an in vivo rat lung model. The vector showed widespread distribution throughout the lung parenchyma, although limited attachment to the airway epithelium. However, gene transfer was not demonstrated despite the use of two reporter genes and additional methods to improve endocytic release. Antisense oligodeoxynucleotide (ODN) therapy for adenovirus infection constituted the other novel therapeutic approach. This study comprised the use of an in vitro biological assay to assess the efficacy of ODNs in modulating adenovirus infection. There was a small, but consistent reduction (p0.005) in adenovirus cytopathic effect associated with an antisense ODN directed to the El A gene of adenovirus 5, compared with the nonsense control ODN. This result suggests a potential therapeutic role for ODNs in adenoviral infection.
... The entry of human Ad into host cells involves the interactions of virus particles with two separate cellular receptors. Initial binding of Ad to target cells is via a fibre protein-mediated attachment to a cellular receptor (Defer et al., 1990), which has recently been identified as a 46 kDa transmembrane protein, called coxsackievirus and adenovirus receptor (CAR) (Bergelson et al., 1997). Virus entry into cells occurs via internalisation into clathrincoated vesicles (Dales and Chardonnet, 1973), and is mediated by an Arg-Gly-Asp (RGD) sequence in the pentone base protein, which binds to cellular integrins and «vP5 (Wickham et al., 1993, Bai et al., 1993). ...
MDCK epithelial cells initially spread in response to hepatocyte growth factor/scatter factor (HGF/SF) followed by the disruption of cell-cell junctions and cell scattering. Their motility involves actin rearrangements mediated by Ras, but how Ras and its downstream effectors also regulate intercellular adhesions has not been established yet. Activation of HGF/SF was shown to induce sustained p42/p44 MAP kinase (MARK) activation, and this was required for spreading, disruption of adherens junctions and subsequent cell scattering as these responses were blocked by the p42/p44 MARK inhibitor PD98059. In addition, activation of phosphoinositide 3-kinase (PI3K) was also required for these responses as they were blocked by its inhibitor LY294002. Disassembly of adherens junctions required both activation of p42/p44 MAPK and PI3K and was a prerequisite for the disruption of desmosomes and tight junctions and subsequent cell scattering In polarized MDCK monolayers, HGF/SF induced a decrease in transepithelial resistance (TER) and loss of intercellular junctions monitored by transmission electron microscopy (TEM) which was PI3K- and p42/p44 MAPK- dependent. To analyse the roles of Rho, Cdc42 and Rac in HGF/SF-induced responses and to allow their expression in over 70% of cells, mutant forms of these GTPases were cloned into adenovirus vectors. Expression of N17RhoA, N17Cdc42, N17Racl and V12Rac1 affected tight junctions as they decreased the TER of MDCK cell monolayers although N17Rac1 transiently increased the TER. N19RhoA and N17Cdc42 dispersed adherens junctions and N17Cdc42 affected the localization of the tight junction protein ZO-1. N17Racl inhibited HGF/SF-induced scattering and disruption of adherens junctions whereas V12Racl enhanced scattering. In conclusion, these results show that HGF/SF- induced scattering and loss of junctions is PI3K- and p42/p44 MAPK-dependent and that these changes are mediated by Ras, Rho and Rac.
... Adenovirus infection can induce CPE with the possibility of cellular lysis [49]. CPE results from the collapse of intermediate filaments network mediated by viral genes products. ...
Human Adenoviruses (HAdVs) are etiological agents of different syndromes such as gastroenteritis, cystitis, ocular, and respiratory diseases, and infection by these viruses may cause alterations in cellular homeostasis. The objective of the study was the proteomic analysis of A-549 cells infected with HAdV-40 using LC–MS. At 30 h of infection, the quantitative analysis revealed 336 differentially expressed proteins. From them, 206 were induced (up-regulated) and 130 were suppressed (down-regulated). The majority of up-regulated proteins were related to energy, cellular organization, stress response, and apoptosis pathways. It was observed alteration of cell metabolism with increase of the glycolytic pathway, β-oxidation, and respiratory chain. Also, the results suggest cytoskeleton reorganization and apoptosis induction. The data can improve knowledge about the replication of HAdV-40 in cell culture considering the proteins related to distinct metabolic pathways induced by viral infection in A-549 cells.
... This is followed by a secondary interaction between virion penton and α v β 3 and α v β 5 integrins, leading to internalization of the virion by clathrin-dependent endocytosis. 12,13 The levels of primary (CAR) and secondary (integrins) receptors present on the cell surface determine the efficiency with which the cell will be infected with adenovirus. 14 After internalization, the acidic environment of the endosome leads to escape of the virion to the cytoplasm. ...
Given the immense potential of recombinant adenoviruses (Ads) as therapeutics for cancer and other genetic disorders, a huge effort has been made in improving the construction and propagation of Ad vectors. This chapter will focus on developments made in the construction and propagation of first generation adenoviral vectors in mammalian cells. The "two-plasmid rescue system" is one of the earliest methods and is still considered the method of choice for the construction of Ad vectors, although it has gone through many refinements. This chapter will walk the readers through development of the two-plasmid rescue system for efficient construction of Ad vectors. Moreover, this chapter discusses different strategies for the construction and propagation of Ad vectors encoding toxic genes that are often troublesome to rescue. Finally, developments in the construction process for generating Ad-based human cDNA libraries are also discussed.
... The studies demonstrating that fiber mediated attachment to the then unidentified receptor did so using the common subgroup C viruses, HAdV-2 and HAdV-5, on cell lines that were permissive to adenovirus infection 16 . Attempts to determine if subgroup B viruses interacted similarly with target cells permissive to subgroup C viruses revealed that subgroup B viruses did not share a common receptor with subgroup C viruses 28,31 . It has since been revealed that CD46, a ubiquitously expressed regulator of complement, serves as a receptor for subgroup B viruses 32 . ...
Vaccines against some of the most pervasive pathogens, including HIV, TB, and malaria, are desperately needed. Available evidence suggests that both central memory and effector CD8+ T cells play a role in mediating protective immunity against many pathogens. Recombinant adenoviruses currently under development as vaccine carriers induce potent and sustained transgene product-specific CD8+ T cell responses. The transgene product-specific CD8+ T cell response remains activated and is delayed in transitioning to central memory. Here we investigate how this response is maintained.
Adenoviral vectors persist and remain transcriptionally active in vivo. We investigated the role that continuous transgene expression plays in maintaining the effector memory-biased transgene product-specific CD8+ T cell response. Dual expression vectors based on AdC7 expressing transgenes that could be regulated temporally were generated. Vectors were unexpectedly unstable in vivo. Alternatively we inhibited the mTOR pathway, which is downstream of the T cell receptor and has recently been implicated in memory development, using a low dose of rapamycin. Rapamycin failed to enhance the quality of the transgene-product specific CD8+ T cells.
Persisting transgene product could maintain the transgene product-specific CD8+ T cell response by continual recruitment of naïve CD8+ T cells, similar to some other persistent viruses. However, in an adoptive transfer model, we demonstrate that the response is primarily maintained by antigen-experienced cells that do not lose function over time.
Pre-existing anti-adenovirus immunity is common, particularly for the human adenoviruses currently in development as vaccine carriers. Here we demonstrate that pre-existing anti-adenovirus neutralizing antibodies accelerate and enhance transgene product-specific CD8+ T cell differentiation into central memory. These cells were highly functional and responded robustly to booster immunization. Importantly, this may be due in part to a reduction the vector's reservoir in T cells.
These data suggest that further study of transgene product expression and its functional impact on immune responses is necessary. Further understanding this impact will allow manipulation of immune responses induced by the adenovirus vaccine platform such that they can be tailored to specific pathogens.
... Ces sucres sont présents en partie terminale des chaînes carbohydrates des glycoprotéines membranaires de la cellule. Les virus du sous-groupe B comme l'HAdV-3 se lie au récepteur membranaire CD46 (Defer et al., 1990;. ...
Adenoviruses are non-enveloped icosahedral viruses. They were found during the 50's and since constitute the huge Adenoviridae family. You can found a large variety of strains able to infect a large variety of hosts from humans to fishes. Fundamental studies on adenoviruses were largely stimulated since the discovery in 1962 by Trentin et al. of their ability to induce tumours in baby hamsters. From the structural point of view, many studies based on electron microscopy were done to understand how the adenovirus capsid is assembled. Since the last fifteen years, progress made in computer science and Electron Microscopy based techniques for 3D image analysis helped scientific community to get a better understanding of the adenovirus capsid structure but many minor proteins were uncertainly localised or totally unknown. Our work is based on Electron Microscopy and 3D image analysis. We obtained 3D models of different adenovirus strains like human, fowl and canine ones where two are below 10 Å resolution. A high resolution 3D reconstruction of Human Adenovirus type 5 helped us to reconstitute a quasi atomic model of the entire capsid and to determine the localisation of minor proteins like IX, IIIa and VIII. We also localised the C terminal part of protein IX from a 3D model of a mutant Human Adenovirus type 2 complexed with a Fab antibody and a canine Adenovirus. Finally, we were interested in the 3D structure of the non mature strain ts1 derived from Human Adenovirus type 2. The “below10Å” 3D reconstruction obtained constitutes infancy into structural understanding of adenovirus maturation and early phase of infection.
... Domains on the knob bind appropriate cell recep− tors [8,9]. They have various additional functions, such as allowing the virus to enter the cell, take part in intracellular transport, and viral growth and accumulation in the cell [13]. Studies of the amino−acid homology of the filaments in AdVs of one or more sub−genera showed that in the case of high homology (above 90%), adenoviruses inter− act with cells similarly and cause similar types of infection in a particular tissue [14]. ...
Adenoviruses (AdVs) are among the main etiological infectious factors that affect immunocompromised patients.They are not potentially dangerous to healthy children, but are a very serious risk for bone marrow and organ recipients. There are about 51 different serotypes of human adenoviruses known so far. They occur generally in the
throat and feces of healthy people. They can be latent in adenoid tissue and kidney, so they can induce disease after many years because of infection reactivation. Adenoviruses are responsible for many disorders. They cause infections of the upper and lower respiratory tracks, alimentary system and urinary track, diseases of the joints,myocarditis, pericarditis and, more rarely, disorders of the central nervous system. Because of the large number of
AdV serotypes, their genomic variations, and their susceptibility to mutation, they present a lot of difficulty in lab−
oratory diagnostics. However, advances in current knowledge create opportunities to introduce new methods toquantify adenoviral infections and monitor therapy efficacy. This article presents current knowledge about adenoviruses and their pathogenicity and information about available methods to diagnose adenoviral infections
... Domains on the knob bind appropriate cell recep− tors [8,9]. They have various additional functions, such as allowing the virus to enter the cell, take part in intracellular transport, and viral growth and accumulation in the cell [13]. Studies of the amino−acid homology of the filaments in AdVs of one or more sub−genera showed that in the case of high homology (above 90%), adenoviruses inter− act with cells similarly and cause similar types of infection in a particular tissue [14]. ...
... Interaction of adenoviral particles with host cell integrins interferes with this adhesion, causing the cell rounding and detachment that lead to early cytopathic effects (CPEs) (Seth, 1999b). The binding of viral particles to the cell surface and some aspects of their internalization into host cells may vary for different serotypes of adenovirus (Defer et al.,1990;Fender et al.,1995). ...
... Both cleavage and phosphorylation of vimentin could lead to rearrangements of the vimentin IF network and its eventual collapse that we observed in MVM-infected cells. For HIV-1 and adenovirus, which induced vimentin rearrangement during infection, it has been shown that it is a result of proteolytic cleavage of vimentin (Belin and Boulanger, 1987;Defer et al., 1990;Shoeman et al., 1990), which then results in the collapse or rearrangements of the vimentin IF network (Honer et al., 1991). In the case of HIV-1, it has been demonstrated that a viral protease cleaves vimentin (Honer et al., 1991;Shoeman et al., 1990). ...
Intermediate filaments (IFs) have recently been shown to serve novel roles during infection by many viruses. Here we have begun to study the role of IFs during the early steps of infection by the parvovirus minute virus of mice (MVM). We found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, we found that vimentin plays an important role in the life cycle of MVM. The number of cells, which successfully replicated MVM, was reduced in infected cells in which the vimentin network was genetically or pharmacologically modified; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles was reduced in cells lacking vimentin. Our data suggests that vimentin is required for the MVM life cycle, presenting possibly a dual role: (1) following MVM escape from endosomes and (2) during endosomal trafficking of MVM.
... The sequence alignment of adenovirus serotypes Ad3 and Ad7 supports this view. Ad3 and Ad7 bind to the same cellular receptor, which is drfferent fiom that bound by Ad5 and Ad2 [39]. Many of the residues which are identical in Ad3 and Ad7, but different fiom thbse in Ad5 and Ad2, are located in the central surface depressions and valleys (Fig. 7). ...
Adenoviral infection begins with the binding of virion to the surface of host cells. Specific attachment is achieved through interactions between host-cell receptors and the adenovirus fiber protein and is mediated by the globular carboxy-terminal domain of the adenovirus fiber protein, termed the carboxy-terminal knob domain.
The crystal structure of the carboxy-terminal knob domain of the adenovirus type 5 (Ad5) fiber protein has been determined at 1.7 A resolution. Each knob monomer forms an eight-stranded antiparallel beta-sandwich structure. In the crystal lattice, the knob monomers form closely interacting trimers which possess a deep surface depression centered around the three-fold molecular symmetry axis and three symmetry-related valleys.
The amino acid residues lining the wall of the central surface depression and the three symmetry-related floors of the valleys are strictly conserved in the knob domains of Ad5 and adenovirus type 2 (Ad2) fiber proteins, which share the same cellular receptor. The beta-sandwich structure of the knob monomer demonstrates a unique folding topology which is different from that of other known antiparallel beta-sandwich structures. The large buried surface area and numerous polar interactions in the trimer indicate that this form of the knob protein is predominant in solution, suggesting a possible assembly pathway for the native fiber protein.
... It has been reported that vimentin rearrangement is important for the viral life cycle, including entry, transport, replication, and egress [16]. A perinuclear condensation of the vimentin IF network observed early in adenovirus infection appears to be a cytological marker for cytoplasmic transit of infectious virions within the infected cells [17]. Risco et al. [18] show that vimentin concentrates in the viral factories of vaccinia virus to participate in vial assembly. ...
Our previous study showed that dengue virus 2 (DENV2) infection induces rearrangement of vimentin into dense structures at the perinuclear area. However, the underlying mechanism of this phenomenon is poorly characterized. In the present work, we found that vimentin and Ser71 phosphorylated vimentin display similar distributions in DENV2-infected cells. DENV2 infection also induced ROCK activation and phosphorylation of vimentin at Ser71 as the DENV2 infection progressed. Furthermore, Ser71 phosphorylation and vimentin rearrangement induced by DENV2 infection were blocked by the ROCK inhibitor Y-27632. In addition, DENV2 led to endoplasmic reticulum (ER) redistribution in the perinuclear region of the host cells, which was partially blocked by pretreatment with Y-27632. Together, these data support indicate that ROCK may have a role in governing regulating vimentin and ER rearrangement during DENV2 infection. We hypothesize that DENV2 infection, via ROCK activation, induces both vimentin rearrangement and ER redistribution around the perinuclear region, which may play a structural role in anchoring DENV2 to replication sites.
... Interaction of adenoviral particles with host cell integrins interferes with this adhesion, causing the cell rounding and detachment that lead to early cytopathic effects (CPE; Seth, 1999b). The binding of viral particles to the cell surface and some aspects of their internalization into host cells may vary for different serotypes of adenovirus (Defer et al., 1990;Fender et al., 1995). ...
Adenoviruses are nonenveloped, double-stranded DNA viruses that infect humans, causing dysentery and respiratory infection. Adenovirus has become a focus of the water treatment community because of its apparent resistance to ultraviolet (UV) disinfection and is the basis for stringent new regulations from the U.S. Environmental Protection Agency regarding UV disinfection of all viruses. Most of the work done so far, however, has involved the use of monochromatic (254 nm) low-pressure UV sources and assay of viral inactivation in cell culture models. Adenovirus is most likely not truly resistant to UV damage but is instead damaged and then repaired in host cells during cell culture infectivity assays. Recent research has shown that newer, polychromatic UV sources are more effective than monochromatic low-pressure UV at inactivating adenovirus. The potential for viral DNA repair in cell culture necessitates the use of alternative assay methods to measure UV disinfection efficiency: these include molecular biology and animal infectivity assays. Research to help clarify the effects of UV on adenovirus should therefore address two major issues not addressed in most studies published so far: the nature of (a) the UV source used to inactivate the virus and (b) the assays used to determine inactivation and characterize viral response. In this review, the authors discuss previous work on UV inactivation of adenovirus as well as present and ongoing work designed to address these two issues.
... This effect was abolished by heat treatment of the virions prior to complex formation. Since heat treatment selectively abrogates adenoviral entry functions without perturbing viral structural integrity (Defer et aL, 1990), it is apparent that the specific internalization functions of the adenovirus comprise a significant component of the gene trans fer capacity of the complexes. Competition for the heterologous epitope on the surface of the chimeric adenovirus by nonlysinated specific monoclonal antibody also attenuated the net gene expression accomplished by the complex. ...
... The normal endosomal acidification process specifically activates viral coat protein domains that trigger disruption of the endosomal membrane, in the case of membranefree viruses such as adenoviruses [77,78], or the fusion of the viral membrane with endosomal membranes in the case of enveloped viruses such as influenza virus [162]. In addition, the viral entry functions have been found to influence the intracellular delivery of other macromolecules163164165. Several groups observed that the presence of adenoviruses during receptor-mediated uptake of macromolecules such as Pseudomonas exotoxin conjugates targeted to the EGF receptor [77,78] or TfR [49] enhanced the entry of the macromolecules into the cell cytoplasm . ...
Transferrin, an iron-transporting serum glycoprotein, is efficiently taken up into cells by the process of receptor-mediated endocytosis. Transferrin receptors are found on the surface of most proliferating cells, in elevated numbers on erythroblasts and on many kinds of tumors. The efficient cellular mechanism for uptake of transferrin has been subverted for the delivery of low-molecular-weight drugs, protein toxins, and liposomes by linkage of these agents to transferrin or to anti-transferrin receptor antibodies. Linkage may be via chemical conjugation procedures or by the generation of chimeric fusion proteins. Transferrin conjugated to DNA-binding compounds (e.g. polycations or intercalating agents) has been successfully used for the import of DNA molecules into cells. High-level gene expression is obtained only if endosome-disruptive agents such as influenza hemagglutinin peptides or adenovirus particles are included which release the DNA complex from intracellular vesicles into the cytoplasm.
... The bootscanning analysis, however, failed to show evidence of recombination, likely because closely related and/or ancestral strains to TMAdV have not yet been identified. Entry of adenoviruses into cells involves an initial attachment of the fiber knob to the cell receptor, followed by internalization via a secondary interaction of the penton base with a v integrins [34,35]. The presence of an RGD motif in the TMAdV penton base implies that the virus uses a v integrins for internalization [35]. ...
Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing
Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3 and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the mRNA and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.
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.
Recombinant viral vectors are an important class of therapeutic gene delivery vehicles. However, because they expose the recipient to foreign proteins, the host immune response is an important consideration in their use in the treatment of human disease. In particular, the toxicity of therapeutically useful viral vectors may limit the dose that can be administered, the duration of transgene expression, and the ability to readminister the vector, all of which may have a significant impact on therapeutic efficacy. Adenovirus, retrovirus, adeno-associated virus (AAV), and herpes simplex virus (HSV) vectors have received the most attention and many clinical trials are under way to test their utility in treating a variety of genetic and acquired disorders. Adenoviral vectors, in particular, are being studied for the treatment of several central nervous system (CNS) and non-CNS diseases (1), and two clinical trials are currently underway using adenovirus vectors to treat gliomas (2). Vector applications can be categorized on the basis of the desired duration of transgene expression and the effect of the immune response on the target cell. For example, correction of such disorders as Parkinson’ s disease (PD) will likely require long-term gene expression and no immune response to the transduced cell. In contrast, viral-based vaccines and anticancer strategies involving immune modulation of brain tumor cells may effectively benefit from short-term gene expression in the context of a significant immune response to the transduced cells. In other clinical applications involving the gene delivered enzyme-prodrug therapies (GDEPT) (3,4) it is not clear how the immune response to the vector will impact on the overall therapy. In our studies, we have found that two successive injections of adenovirus expressing the HSV thymidine kinase (HSVTK) gene in conjunction with systemic ganciclovir (GCV) has been well tolerated. Patients have received up to 109 pfu of virus with concomitant administration of large doses of glucocorticoids with no apparent toxicity to date (5; J. Alavi, K. Judy, and S. Eck, University of Pennsylvania, unpublished observations).
Adenovirus vectors have shown seductive promise as molecular biology tools and are being proposed for a number of clinical gene delivery applications. We will review the properties of adenovirus vectors that are responsible for the great interest in their use. We will discuss a number of the limitations of the current vectors and describe some of the strategies that are being used to improve these vectors.KeywordsAdenovirus VectorRecombinant AdenovirusYeast Artificial ChromosomeAdenovirus TypeHelper VirusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields.
Genes are the basis of much human disease, whether they are the defective genes involved in a hereditary disease, the genes of a pathogen dedicated to taking advantage of a host’s physiology for their own ends, or the wayward genes of a cancer promoting uncontrolled cell division. The rapid advances in genetics since the early 1970s, notably the development of recombinant DNA techniques, have allowed the isolation and manipulation of genetic material and permitted the identification of some of the various genes involved in human disease.
Die Restenoserate nach koronarer Ballonangioplastie (PTCA) [38] und anderen perkutanen Interventionen am Koronarsystem (PCI; Rot-ablation) [49,173], Atherektomie [7,124,193],Laserangioplastie [115,172] ist trotz intensiver experimenteller und klinischer Forschungsarbeiten weiterhin hoch. Die Verwendung von Stents hat nach großen klinischen Studien die Restenoserate von etwa 40-50% auf etwa 20-30% gesenkt [20,61,188,189], eine Reihe anderer Ansätze zur Restenoseprophylaxe wie die Brachytherapie [208,210] sind derzeit in der klinischen Evaluierung (siehe S. 420-424 Status der Rezidivprophylaxe). Die hierdurch notwendigen Reinterventionen stellen bisher angesichts von jährlich mehr als einer Million Interventionen weltweit nicht nur eine große Belastung für die betroffenen Patienten dar, sondern besitzen auch erhebliche gesundheits-ökonomische Bedeutung. D a bisher keine befriedigende Lösung des Problems erreicht worden ist, wurden in den letzten Jahren auch innovative experimentelle Ansätze zur Restenoseprophylaxe evaluiert.
Gene transfer to eucaryotic cells may be accomplished by capitalizing on endogenous cellular pathways of macromolecular transport. In this regard, gene transfer has been accomplished via the receptor-mediated endocytosis pathway employing molecular conjugate vectors (Wu and Wu, 1987; Wu et al., 1989; Wagner et al., 1990; Wagner et al., 1991b; Cotten et al., 1990; Curiel et al., 1992a; Huckett et al., 1990; Rosenkranz et al., 1992). The molecular conjugate is a synthetic bifunctional agent employed to serve two functions: binding of the heterologous DNA to form a conjugate-DNA complex, and attachment of the conjugate-DNA complex to the target cell to facilitate internalization. The initial step of binding the heterologous DNA to form a conjugate-DNA complex is mediated through a domain of the conjugate comprised by a polycation amine, such as polylysine. This electrostatic interaction between the negatively charged DNA and the positively charged DNA-binding domain serves not only to bind the DNA, but also to condense it into a compact toroid structure (Wagner et al., 1991a). As the ligand is covalently linked to the polylysine DNA-binding domain, after binding of the DNA, at least a portion of the ligand is presented on the surface of the complex, free to interact with its cognate receptor. The attachment of the conjugate-DNA complex to the target cell is then mediated through the specificity of the ligand domain for its corresponding receptor. Thus, after the conjugate-DNA complex accomplishes attachment, it is internalized through the corresponding cellular transport pathway.
Objective: To study the effect of human cytomegalovirus (HCMV) on expressions of K8 and K18 in duct epithelial cells of salivary gland. Methods: The expressions of immediate early antigen of HCMV, K8 and K18 were detected by immunohistochemistry staining in tissues embedded in paraffin of carotid cytomegalic inclusion disease (PCID). Results: Cytomegly bearing inclusion appeared in duct epithelium of PCID. DDG9/CCH2 antigen of HCMV was expressed in cytomegly bearing inclusion. K8 was negative in these cytomegly while K18 was intensively positive. Conclusion: It is suggested that breaking down of K8 be induced in parotid duct epithelial cells infected by HCMV and that up-regulation of K18 may be a reactive change. Keratin network in simple epithelium functions to impart mechanical integrity to cells.
Viruses have evolved highly efficient mechanisms to transfer their genetic material to target cells (Kielian and Jungerwirth 1990; Hoppin and Scheid 1980; Helenius 1992). These mechanisms have been capitalized on in the design of recombinant viral gene transfer vectors. The central strategy for the design of these agents has involved the incorporation of heterologous DNA sequences into the genome of the parent virus. Thus, in the process of delivering its endogenous nucleic acid sequences, the virion would also deliver the incorporated heterologous sequences. Using this basic paradigm, a variety of both DNA and RNA recombinant virion species have been developed that have proved to be of great utility for gene therapy applications (Berkner 1988; Eglitis and Anderson 1988).
Although human autologous B cells represent a promising alternative to dendritic cells (DCs) for easy large-scale preparation, the naive human B cells are always poor at antigen presentation. The safe and effective usage record of Human adenovirus type 7 (HAdV7) live vaccines makes it attractive as a promising vaccine vector candidate. To investigate whether HAdV7 vector could be used to induce the human B cells cross-presentation, in the present study, we constructed the E3-defective recombinant HAdV7 vector encoding green fluorescent protein (GFP) and carcinoembryonic antigen (CEA). We demonstrated that naive human B cells can efficiently be transduced, and that the MAPKs/NF-κB pathway can be activated by recombinant HAdV7. We proved that cytokine TNF-α, IL-6 and IL-10, surface molecule MHC class I and the CD86, antigen-processing machinery (APM) compounds ERp57, TAP-1, and TAP-2. were upregulated in HAdV7 transduced human B cells. We also found that CEA-specific IFNγ expression, degranulation, and in vitro and ex vivo cytotoxicities are induced in autologous CD8(+) T cells presensitized by HAd7CEA modified human B cells. Meanwhile, our evidences clearly show that Toll-like receptors 9 (TLR9) antagonist IRS 869 significantly eliminated most of the HAdV7 initiated B cell activation and CD8(+) T cells response, supporting the role and contribution of TLR9 signaling in HAdV7 induced human B cell cross-presentation. Besides a better understanding of the interactions between recombinant HAdV7 and human naive B cells, to our knowledge, the present study provides the first evidence to support the use of HAdV7-modified B cells as a vehicle for vaccines and immunotherapy.
Previous studies of egg drop syndrome virus (EDSV) are restricted to serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV are poorly understood. Therefore, we aimed to explore the entry pathway of EDSV into duck embryonic fibroblast (DEF) cells as well as the infection characteristics and proliferation of EDSV in primary DEF and primary chicken embryo liver (CEL) cells. Transmission electron microscopy revealed that the virus triggered DEF cell membrane invagination as early as 10min post-infection and that integrated endocytic vesicles formed at 20min post-infection. The virus yield in EDSV-infected DEF cells treated with chlorpromazine (CPZ), sucrose, methyl-β-cyclodextrin (MβCD), or NH4Cl was measured by quantitative real-time PCR. Compared with the mock treatment, CPZ and sucrose greatly inhibited the production of viral progeny in a dose-dependent manner, while MβCD treatment did not result in a significant difference. Furthermore, NH4Cl had a strong inhibitory effect on the production of EDSV progeny. In addition, indirect immunofluorescence demonstrated that virus particles clustered on the surface of DEF cells treated with CPZ or sucrose. These results indicate that EDSV enters DEF cells through clathrin-mediated endocytosis followed by a pH-dependent step, which is similar to the mechanism of entry of human adenovirus types 2 and 5.
Copyright © 2015. Published by Elsevier B.V.
Adenoviruses are promising vectors for human cancer gene therapy. However, the extensively used adenoviruses serotypes 2 and 5 (Ad2 and Ad5) from species C have a major disadvantage in being highly prevalent; thus, most adults have an immunity against the two viruses. Furthermore, the expression of coxsackievirus and adenovirus receptors for Ad2 and Ad5 varies in different cells. This study aims to identify adenovirus serotypes with specific tropism for endothelial cells and epithelial tumour cells. Comparison of the binding affinities of Ad31, Ad11, Ad5, Ad37, Ad4 and Ad41, belonging to species A-F, respectively, to established cell lines of hepatoma (HepG2), breast cancer (CAMA and MG7), prostatic cancer (DU145 and LNCaP) and laryngeal cancer (Hep2), as well as to endothelial cells (HMEC), was carried out by flow cytometric analysis. Ad11 from species B showed markedly higher binding affinity than Ad5 for the endothelial cell line and all carcinoma cell lines studied. Ad4 showed a specific binding affinity for hepatoma cells and laryneal carcinoma cells. The ability of Ad11, Ad4 and Ad5 to be expressed in hepatoma, breast cancer and endothelial cell lines was studied by immunostaining and (35)S-labelling of viral proteins in infected cells. Ad11 and Ad4 manifested a higher proportion of infected cells and a higher degree of hexon expression than Ad5.
The research on intracellular trafficking of adenovirus has been described mainly through observations of subgroup C adenoviruses in transformed cell lines. The basic elements of the trafficking pathway include binding to receptors at the cell surface, internalization by endocytosis, lysis of the endosomal membrane, escape to the cytosol, intracellular trafficking along microtubules, nuclear pore docking, and viral genome translocation into the nucleus. More than 80% of the adenovirus genome is delivered to the nucleus in a highly efficient manner in approximately 1 h. However, exceptions to this trafficking pattern have been noted, including: variations based on target cell type, cell physiology, and adenovirus serotype. This review summarizes mechanism of adenovirus infection pathway and intracellular trafficking, providinging a foundation for the development of clinical adenoviral vector.
Strategies have been developed to accomplish gene delivery via the receptor-mediated pathway employing molecular conjugate vectors (1-13). As cells possess endogenous pathways for internalization of macromolecules, the utilization of these pathways for the purpose of DNA delivery represents a strategy that potentially allows certain practical advantages. In this regard, these cellular internalization pathways can be highly efficient. For example, internalization of the iron transport protein transferrin can be on the order of thousands of molecules per minute per cell (14,15). These pathways thus represent a potentially efficient physiologic method to transport DNA across the cell membrane of eukaryotic cells. To accomplish gene transfer via receptor-mediated endocytosis, a vehicle must be derived that allows DNA entry into these cellular pathways. For this purpose, molecular conjugate vectors have been derived. These vector agents consist of two linked functional domains: a DNA-binding domain to transport the DNA as part of the vector complex, and a ligand domain to target a cellular receptor that allows entry of the conjugate-DNA complex into a receptor-mediated endocytosis pathway. For incorporating DNA into the complex for gene delivery, binding must be achieved in a nondamaging, reversible manner. For this linkage, an electrostatic association between the binding domain and the nucleic acid is accomplished. To achieve this, the DNA binding domain is comprised of a polycationic amine, such as poly(L)lysine. This can associate with the negatively charged DNA in an electrostatic, noncovalent manner. To achieve entry of the complex through a receptor-mediated pathway, a ligand for the target cell is utilized. The ligand domain is covalently linked to the polylysine to create the molecular conjugate vector. The ligand domain may be a native or synthetic cell surface receptor ligand, an antireceptor antibody, or other agent that allows specific association with target cell membranes.
Despite development in surgical techniques, chemotherapy and radiotherapy,
most malignancies of the central nervous system are still devastating
tumors with a poor prognosis. For example, median survival of
patients with malignant gliomas (astrocytoma, oligodendroglioma or
mixed rype) is roughly 12 months and only 5 % of the patients survive
more than 5 years after diagnosis. Fifty % of astrocytomas are ryped
as glioblastoma multiforme, the most malignant form of glioma.
Glioblastoma account for 15-23 % of all intracranial tumors and have a
very poor median survival of 6 months with conventional therapy.
Metastases account for 15-30 % of all intracranial neoplasm's and develop
in 25 to 30 % of all cancer patients. The overall median survival
time after surgery followed by radiation therapy in solitary metastatic
lesions ranges from 9 to 23 months, depending on the rype of primary
cancer. The prognosis in patients with multiple metastases, however,
is much worse. Lepto-meningeal metastases from solid tumors
are of increasing importance in neuro-oncology, because of the increasing
frequency and the severe neurologic disabiliry it causes. About
0.8 to 8 % of patients with cancer develop leptomeningeal metastases and
median survival in these patients after radio- and chemotherapy ranges
from 7 to 24 weeks.
As the number of products being sold online increases, it is becoming increasingly difficult for customers to make purchasing decisions based on only pictures and short product descriptions. Thus, customer reviews, particularly the text describing the features, comparisons and experiences of using a particular product provide a rich source of information to compare products and make purchasing decisions. Especially, all kinds of reviews from various people have different degree of impact on a buyer, that is, we tend to believe our friends who always make right decisions than others. In this paper, we present an individual feature-based product ranking technique that mines thousands of customer reviews. By grouping users into unfamiliar users and familiar users according to the fact whether the client has almost always right ideas as far as one has concerned we attach different weights to them based on the friend ranking list. Friends on the top of the list are expected to be more reliable than the rest. After founding the client's friend set{F_j, S_k}, we extract crucial information from users' reviews. By realizing key words in a sentence, we classify comments into 4 representative sentences-Active Direct sentence(AD), Inactive Direct sentence(ID), Active Indirect sentence(AI), and Inactive Indirect sentence(II). Afterwards, we construct a weighted graph considering the product weight itself and the edge between every 2 relevant products, using ratios AD/ID and ID/II. The last step is that the client ranks search result with the average reliabilities of himself with respect to reviews of specific feature. Through calculation, we have a weighted score list, helping the client make purchase intentions.
Clinical application of gene therapy for patients who have inflammatory bowel disease or short bowel syndrome will require the development of new strategies to improve the efficiency of small intestinal gene transfer. Previously, the authors developed a method for adenoviral-mediated small intestinal gene transfer in vivo in neonatal and adult mice. The present study evaluates the hypothesis that the integrins alpha(v)beta3 and alpha(v)beta5, the secondary receptors for adenoviral internalization, play a facilitative role in neonatal murine adenoviral-mediated small intestinal gene transfer.
Immunohistochemical techniques identified the integrin alpha(v)beta3 and the integrin subcomponents alpha(v), beta3, and beta5 in neonatal and adult small intestine. The effects of integrin receptor antagonists on transgene expression was also studied in our neonatal model of adenoviral-mediated small intestinal gene transfer in vivo.
Gene transfer was significantly decreased by the addition of integrin receptor antagonists versus control peptide. Integrin alpha(v)beta3 and integrin subcomponent alpha(v), beta3, and beta5 are expressed in neonatal and adult small intestine. Integrin antagonists administered simultaneously blocked efficient adenoviral-mediated neonatal small intestinal gene transfer in vivo compared with control peptide.
Strategies to upregulate integrin expression may improve adenoviral-mediated small intestinal gene transfer.
The efficacy of oncolytic virotherapy is influenced by the interactions between the tumour, virus and host immunity. The first generation of oncolytic adenoviruses, based on serotype 5 (Ad5), has achieved limited success in clinical trials. Its shortcomings include the downregulation and inaccessibility of its receptor, the Coxsackie and adenovirus receptor (CAR) in cancer cells, high prevalence of neutralising antibodies and hepatotoxicity. In contrast, Ad11 binds to CD46 and other receptor(s) but its potential as an oncolytic virus remains to be explored.
A panel of human cancer cell lines were found to express higher levels of CD46 than CAR. However, not all cell lines were more sensitive to Ad11-mediated cytotoxicity in vitro compared to Ad5. Treatment of Ad5-insensitive PC-3 human prostate cancer xenografts with Ad11 resulted in significant reduction in tumour growth, but not Ad11-insensitive MIA PaCa-2 human pancreatic cancer xenografts. Virus attachment and nuclear entry of Ad11 were significantly better than Ad5 even in cells that were insensitive to Ad11 killing. In these cells, however, Ad11 E1A mRNA levels were much lower than those of Ad5, producing a negative effect on viral DNA amplification, structural protein synthesis, progeny production and cell killing. Cells that were sensitive to Ad11 cytotoxicity showed higher levels of E1A mRNA.
The region upstream of Ad5 E1A demonstrated higher transcription-enhancing activity than the corresponding region of Ad11. Two Ad11 mutants were constructed in which E1A was under the control of the Ad5 E1A promoter and enhancer-promoter, respectively. With the latter virus, improved oncolytic potency was observed. It was superior to Ad11 and also to Ad5 in many cancer cell lines, and was as effective as Ad5 in the MIA PaCa-2 xenograft model. Therefore, Ad11 with the Ad5 E1A enhancerpromoter should be used as a backbone for the future development of potent and tumour-specific oncolytic Ad11 mutants.
Many types of viral infection produce a transient permeabilization of the Infected cell (see
refs.
1 and 2; reviewed in refs.
3 and 4). A large improvement in transfection efficiency was obtained when this phenomenon was applied to receptor-mediated gene
delivery (5). A number of permeabilizing agents have now been used to enhance gene transfer, but adenovirus is by far the most potent
(6,7). The augmentation of DNA delivery by adenovirus particles is primarily due to enhancement of cytoplasmic entry of DNA; however,
viral components may also serve additional functions that promote transfection, such as generating alterations in the cytoskeleton
of the transfected cell (8) or in promoting nuclear entry (9,10). The most efficient varrations of this method employ some form of linkage between the adenovirus and the plasmid DNA, thus
ensuring that each viral entry event is linked to plasmid DNA entry. There are now a number of methods of linking DNA to carrier
adenovirus. A versatile method involves biotinylating the adenovirion and linking the plasmid DNA with a streptavidin-polylysine
bridge (6). Other methods include direct chemical linkage between virus and polylysine (11–14), as well as an enzymatic linkage using transglutaminase (15,16). The drawbacks with the direct virus-polylysine linkage methods are the generation of a fixed virus-to-polylysine ratio
(limiting titration possibilities), and the storage difficulties of polylysine-adenovuus that necessitate the use of high
tonic strengths. A coupling technique using antibody-polylysine binding to an adenovirus epitope has been described that allows
a greater degree of flexibility in virus-polylysine ratios (17, but requires both an epitope-tagged virus and a special antibody-polycation
conjugate.
The fiber protein of adenovirus mediates the interaction of adenovirus with cell membrane receptors. We have produced the Ad3 fiber protein in the baculovirus expression system. Biochemical, morphological and functional analyses showed that the recombinant fiber was properly folded and functionally competent. The specific binding of Ad3 virus to two HeLa membrane proteins of 130 and 100 kDa was demonstrated with an overlay protein binding assay. In the same assay, Ad3 fiber only recognized the 130-kDa protein. Divalent cations seemed to be important for the interaction of both virus and fiber with these proteins.
We have identified an adenovirus type 5(Ad5) early gene function located in early region 1 which is required for the production of early cytoplasmic mRNAs corresponding to early regions 2, 3, and 4. Mutant dl312 (lacks the segment between 1.5 and 4.5 map units) grows as well as wild-type virus in 293 cells (Ad5-transformed human embryonic kidney cells), but its growth is severely restricted in HeLa cells. We detect no viral RNAs in the cytoplasm of dl312-infected HeLa cells. Viral RNA sequences are present, however, in dl312-infected HeLa cell nuclei.
A temperature-sensitive mutant of human adenovirus type 2, ts112, was isolated and characterized, ts112 was blocked in a late function required for virus maturation. At restrictive temperature, it accumulated light precursor particles that were able to mature into infectious virions upon temperature shift-down. Use of a mild extraction procedure and a reversible fixation by a cleavable diimido ester permitted the isolation and analysis of these labile intermediates in the adenovirus assembly. These accumulated particles had a sedimentation coefficient of about 600S and a buoyant density of 1.315 g/cm3 in CsCl. They contained a DNA fragment of 7--11S and two nonvirion proteins having molecular weights of 50,000 (50K) and 39.000 (39K), respectively. They resembled in composition and morphology the light intermediate particles found in wild-type adenovirus 2, which were identified as precursors of heavy intermediates, preceding the young virions. The ts112 lesion was apparently located at the exit of either the 50K and/or 39K proteins and at the entry of viral DNA.
Cellular repair of DNA damage due to lethal gamma irradation was studied to reveal differences between strains and cell cycle stages that are otherwise difficult to detect. Cycling and metaphase-blocked cultures of normal fibroblasts and carcinoma cells were compared for repair of gamma sites (gamma radiation-induced nicks, breaks, and alkali-labile sites in DNA) at supralethal exposures ranging from 7 to 150 krad 137Cs radiation and at postirradation incubations of 20-180 min. Fibroblasts from normal human skin or lung repaired gamma sites efficiently when cycling but did not repair them when blocked at mitosis. Bladder (253J) or lung (A549) carcinoma cells, unlike normal fibroblasts, repaired gamma sites efficiently even when blocked at mitosis. Whether the above differences in DNA repair between cell cycle stages and between strains result from differences in chromatin structure (cis effects) or from differences in the nuclear enzymatic environment (trans effects) could be resolved by placing an inert, extrachromosomal DNA molecule in the cell nucleus. Specifically, cis effects should be confined to the host chromosomes and would not be detected in the inert probe whereas trans effects should be detected in host chromosomes and inert probe DNA alike. Indeed, we found a suitable DNA molecule in the adenovirus deletion mutant dl312, which does not proliferate in the absence of E1A complementation. Gamma sites in 32P-labeled adenovirus dl312 DNA were repaired efficiently in all hosts, regardless of mitotic arrest. Failure of mitosis-arrested fibroblasts to repair gamma sites was therefore due to a cis effect of chromatin organization rather than to a trans effect such as repair enzyme insufficiency. In sharp contrast, chromosomes of mitotic carcinoma cells remained accessible to repair enzymes and nucleases alike. By means of these new tools, we should get a better understanding of higher-order chromatin management in normal and cancer cells.
Regions of the adenovirus type 5 early region 1A (E1A) proteins that are required for transformation were defined by using a series of deletion mutants. Deletion mutations collectively spanning the entire protein-coding region of E1A were constructed and assayed for their ability to cooperate with an activated ras oncogene to induce transformation in primary baby rat kidney cells. Two regions of E1A (amino acids 1 to 85 and 121 to 127) were found to be essential for transformation. Deletion of all or part of the region from amino acids 121 to 127 resulted in a total loss of transforming ability. An adjacent stretch of amino acids (residues 128 to 139), largely consisting of acidic residues, was found to be dispensable for transformation but appeared to influence the efficiency of transformation. Amino acids 1 to 85 made up a second region of the E1A protein that was essential for transformation. Deletion of all or part of this region resulted in a loss of the transforming activity. Even a mutation resulting in a single amino acid change at position 2 of the polypeptide chain was sufficient to eliminate transformation. Deletion of amino acids 86 to 120 or 128 to 289 did not eliminate transformation, although some mutations in these regions had lowered efficiencies of transformation. Foci induced by transformation-competent mutants could be expanded into cell lines that retained their transformed morphology and constitutively expressed the mutant E1A proteins.
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.
When KB cells were labeled with either 51Cr (1 microCi/ml) or [35S]methionine (5 microCi/ml) and treated with 10 micrograms/ml of adenovirus type 2 (Ad2) at pH 6.0 for 60 min at 37 degrees C, about 25% of the cell-associated 51Cr and 5% of the [35S]methionine were released into the medium. The 51Cr was mainly associated with molecules of 1500 Da or less. When KB cells were labeled with either [3H] choline, alpha-[3H]aminobutyric acid, or [3H]deoxy-2-fluoro-D-glucose and exposed to Ad2, these molecules were released in amounts much higher than 51Cr. The Ad2-dependent release of choline was found to be dependent on Ad2 concentration, with maximum release (nearly 60%) at 10 micrograms/ml of Ad2, on the length of the incubation with Ad2, with maximum release at about 90 min, and on the medium pH with maximum activity at pH 6.0 to 6.5. Greater than 95% of the choline released was water-soluble and identified as choline phosphate. Less than 5% of the choline released was associated with lipids, and none was released as a phospholipid vesicle or micelle. The ability of Ad2 to release choline was abolished by incubating Ad2 for 10 min at 45 degrees C, whereas the binding of Ad2 to the cells was not affected. Fetal calf serum also blocked Ad2-dependent choline release.
HeLa or KB cells each contain around 10(4) receptor sites for adenovirus type 2. These are inactivated by treatment of live cells with subtilisin. The receptor activity of the enzyme-treated cells is regained after 4 to 8 hr of incubation in complete medium. A technique that utilizes the difference in buoyant density between free virus and virus-receptor complex was developed to demonstrate receptor activity. Cellular fractionation revealed that receptors were confined mainly to the plasma membrane fraction and that negligible receptor activity could be demonstrated in enzyme-treated cells. Subtilisin probably did not penetrate the cell membrane; thus, the receptors are limited to the cell surface. Purified fiber of the virion completely prevents attachment of adenovirus types 2 and 5 to receptor sites at a ratio of 10(5) protein molecules per cell. Adsorption studies indicate that 10(5) to 10(6) receptor sites are available for the structural protein. The fiber does not affect attachment of poliovirus type 1.
Schmltz, H., R. Wlgand (Instltut für Hygiene, U. Kliniken, D-6650 Homburg/ Saar, FRG), and W. Helnrich. Worldwide epidemiology of human adenovlrus infections. Am J Epidemiol 1983; 117: 455–66.
Approximately 25, 000 reports to WHO from 1967 to 1976 on isolations of adenovirus 1 to 31 showed an absolute frequency of Ad2, 1, 7, 3, 5, 6, 4, 8, and other species, in decreasing order. The relative frequency was analyzed by the X² method; only significant deviations (p < 0.05; in many cases p < 0.001) from chance distribution were taken into account. To avoid small numbers, the three adenovirus species of subgenus A (Ad12, 18, 31) and several species of subgenera B and D were analyzed as one group. The incidence of Ad8, 7 and 19, less so of Ad3 and 4, showed a periodicity over the years. No definite seasonal incidence pattern was found for the countries of the Northern Hemisphere. The Southern Hemisphere showed a higher incidence of Ad4 and 7, and subgenus B (without Ad3 and 7), and a lower incidence of Ad6, whereas subgenus A, Ad8, and 19 were rarely reported. Highly significant age predilections were subgenus A for infants, subgenus C for infants and small children, Ad3 for school children, Ad7 for school children and adults, Ad4, 8, and other species of subgenera B and D for adults. A predilection for males was observed In all species of subgenera B and C, and in Ad4 and 19. The association between virus infection and clinical syndromes was analyzed for children up to age 14 years and for adults separately. Subgenus C viruses, mainly Ad2, occurred frequently in Infections of the lower respiratory and gastrointestinal tract In both children and adults. An association with disease of the central nervous system, fever, and cardiovascular disease was seen in adults only. Ocular infections in both children and adults were associated with Ad3; only children showed an association with upper respiratory tract disease and fever. Ad7 was frequent in ocular disease and fever in children, and in respiratory infections in adults. For Ad4, no organ specificity was found. Ocular infections in both children and adults were associated with Ad8; only children showed an association with upper respiratory tract disease. Subgenus A was associated with gastrointestinal disease in children. The hypothesis that all the adenovirus species from one subgenus may have similar epidemiologic and pathogenic properties appears to be largely true for subgenus C viruses. In subgenus B, many differences were found between Ad3 and 7, and other viruses of subgenus B.
Historically, two divergent views have evolved about the specificity of animal virus attachment to susceptible host cells. One emphasizes the absence of precise structural requirements for virus-cell interaction (Dales, 1973) and the second proposes specific cellular receptor sites in the plasma membrane which recognizes one or several of the virus attachment proteins (VAP) (Lonberg-Holm and Philipson, 1980). The narrowly defined host range of bacteriophages and the identification of their receptors as cellular components indicate that the interaction of the phages with the host cells resembles the lock and key interaction between enzyme and substrate. Although animal viruses can adsorb non-specifically to artificial membranes or inert supports (Boche and Quilligan, 1966; Huang, 1974) it has been clearly demonstrated for enteroviruses that cells that lack receptors are also virtually resistant to infection by intact viruses (Holland et al., 1959; McLaren et al., 1960; Soloviev et al., 1968), although they are susceptible to infection with extracted viral nucleic acids (Holland et al., 1959; McLaren et al., 1960). There is also considerable species specificity for virus infection for several non-enveloped viruses, e.g. picornaviruses (Lonberg-Holm and Philipson, 1974), adenoviruses (Philipson et al., 1975), and parvoviruses (Linser et al., 1977). In addition studies with hybrid cell lines have established that there are genes for specific cellular binding sites for several picornaviruses (Green et al., 1971).
Non-enveloped animal viruses, in most cases, have icosahedral symmetry. Therefore, they have at least 12 identical sites for interaction with receptors on host cells. The multiple subunit structure of the virion capsid leads not only to multivalent receptor bonding, but may also confer allosteric properties to the virion. In this chapter, we will discuss structural features of primarily two families of non-enveloped viruses. The native capsids of many picoraviruses undergo major conformational changes when disrupted and their individual subunit polypeptides cannot be isolated easily in their ‘native’ condition or assigned separate biological functions. Adenoviruses, in contrast, provide an example where isolated capsid subunits retain activity, and one of these, the fiber, has been identified as the virus attachment protein which recognizes cellular receptors, while other subunits have other biological functions. Other non-enveloped viruses will be discussed briefly in relationship to these two families.
One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) could support acidification. The ATP-dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP-dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.
The effect of a number of drugs and culture conditions on the entry into cells of a strain of poliovirus 1 (Brunende) was tested. The cells were exposed in the dark to light-sensitive, neutral red-containing virus, in the presence of the drug to be tested. Then the cells were exposed to light, transferred to normal medium, and incubated overnight. Cytopathogenic effect was measured as inhibition of [3H]leucine incorporation. Compounds that dissipate proton gradients across membranes, like monensin, protonophores, and amines, and compounds that inhibit the acidification process, such as N,N'-dicyclohexylcarbodiimide (DCCD) and tributyltin, inhibited the entry of virus, but not virus binding. This was also the case with metabolic inhibitors that deplete cells for ATP. The same compounds also inhibited the cell-induced alteration of the virus particles. When cells with surface-bound virus were exposed to low pH, the virus entered efficiently, even in the presence of monensin and DCCD. The results indicate that acidification somehow facilitates the entry of the virus RNA into the cytosol and that under normal conditions the entry occurs from intracellular acidic vesicles.
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.
Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.
Infection of mouse L cells with vaccinia virus leads to a modification in the permeability of the cell membrane. This modification permits the entry of the translation inhibitor hygromycin B (MW 550). On the basis that some inhibition of protein synthesis is also observed in the presence of the protein toxin α-sarcin (MW 16,800), it seems likely that macromolecules can also cross the membrane in vaccinia virus-infected cells. The modification of the cell membrane is observed very early during infection, in the first hour of viral adsorption. After this time the cell membrane gradually reseals, restoring its normal permeability characteristics. The multiplicities of infection needed to observe these modifications in permeability are rather low; the presence of 3 PFU/cell (150 physical particles/cell) are enough to promote the entry of the inhibitor hygromycin B. Ultraviolet (uv)-inactivated vaccinia virus (17,280 ergs/mm2 promotes the increase in membrane permeability. Moreover, the presence of actinomycin D (25 μg/ml) and cordycepin (100 μg/ml) did not inhibit the passage of hygromycin B suggesting that a virion component is responsible for this phenomenon. That an intact virion is required to permeabilize L cells, is supported by the findings that neither purified core particles, nor virion surface proteins can induce the inhibition of translation by hygromycin B. Since interferon treatment appears to induce chemical, physical, morphological, and immunological alterations in the cell surface, the effect of interferon on changes in membrane permeability following vaccinia virus infection was investigated. We find that the permeability of the cell membrane is enhanced in interferon-treated, vaccinia virus-infected cells when compared to untreated, infected cells.
Because of the importance of quantitative determination of protein in the research laboratory as well as in the food and feed industries (1), search for the ideal method continues unabated after many years. Methods available include nitrogen determination (Kjeldahl (2) and Dumas (3)), hydrolysis of the protein, derivatization of the amino acids with phthalaldehyde and fluorescence determination (4), determination of bound or free lysine (5) or glutamate (4), and the Lowry (6), biuret (7) dye-binding (8–11) turbidity (12) and spectral methods (13). With the exception of the spectral methods, the methods involve destruction of the sample.In this paper we report the use of difference in absorbance between 235 and 280 nm for determination of protein concentration.
According to folk tradition, the symptoms of influenza can be relieved by spending a night in the stables. The scientific basis for this belief is obscure, but it may have something to do with the air being rich in ammonia. Ammonia and other weak bases which accumulate in lysosomes have been found to inhibit infection by many animal viruses. Studies using these compounds are now advancing our understanding of the processes by which enveloped animal viruses enter and infect cells.
THE specific receptors for several non-enveloped viruses are present on cells in only limited numbers (1 × 104-10 × 104), and can therefore be saturated with excess virus1-5. An excess of inactivated poliovirus type 1 inhibits the attachment of infectious poliovirus of all 3 serotypes, but not the attachment of other viruses, including B Coxsackie viruses6. Most of the B Coxsackie viruses probably share the same receptor7, but Coxsackie viruses B1 and B3 do not compete for the poliovirus receptor1. Adenovirus 2 and 5 also appear to share the same receptor, since their attachment to host cells is blocked by an excess of the adenovirus type 2 fibre protein, which seems to be the receptor-recognising portion of the virus particle2. A number of other adenoviruses may also belong to the same receptor family since soluble antigens block their attachment to erythrocytes8. We here report results which permit the construction of a `receptor map' for a number of non-enveloped viruses.
This chapter discusses the processes of recognition, adsorption, and penetration of animal viruses, as well as the changes induced in the infecting viruses and the “infected” plasma membrane on a time scale of minutes rather than hours or days. The first step in the virus-cell interaction is the attachment of the virus to a specific cell receptor. The receptors that bind viruses are located on the body of the cell as well as on microvilli, but they seem to be absent from the inner structures of the cell. The first step in the virus-cell interaction is the attachment of the virus to a specific cell receptor. The attachment of the viruses to these sites is the first step in the process of their penetration and infection. Picorna virus receptors on the cell membrane are sensitive to chymotrypsin, but can be regenerated following enzyme degradation provided there is protein synthesis on the cell. The state of fluidity of the cellular membranes is a factor not only in the cell's social behavior and in various secretory processes, but also in the interaction of viruses with cells and in the maturation of enveloped viruses. Extracellular materials can enter a cell either by a specific transport mechanism, by passive flux or by endocytosis and fusion. An important and fascinating step occurring early in most cytocidal viral infections is the shut off macromolecular syntheses in the infected cells-predominantly of RNA and protein. A very specialized and complicated defense mechanism of cells to prevent viral infection involves interferon.
Three different approaches were used in an attempt to characterize the KB-cell receptor for adenovirus: affinity chromatography, immunoadsorption and cross-linking with a cleavable bifunctional reagent. The first system used an affinity gel consisting of adenovirus-fibre projection linked to Sepharose matrix by an intermediate bis(aminopropyl)amine arm, the amino groups of the fibre ligand being preserved by prior citraconylation. The second system consisted of adenovirus complete penton capsomere attached to anti-(penton base) antibody and cross-linked to polyacrylamide particles with glutaraldehyde. In this latter affinity model, the penton-fibre projection was appropriately oriented outwards, as in the virus particle. Both affinity systems permitted isolation from a KB-cell plasma-membrane extract of fibre-binding and penton-fibre-binding protein material, which inhibited adenovirus attachment. The penton-immunoadsorbent appeared more efficient and more specific than the affinity column of fibre-bis(aminopropyl)amino-Sepharose gel in specific activity of inhibition of adenovirus attachment. The third method consisted of reversibly cross-linking KB-cell receptor proteins with adenovirus particles by means of a cleavable di-imidoester and isolation of the complexes by sucrose-density-gradient centrifugation. Polypeptide analysis on sodium dodecyl sulphate/polyacrylamide gel of labelled KB-cell surface proteins, selected by the different procedures, showed that three major protein subunits of 78000, 42000 and 34000mol.wt. were common to the three selection systems. A possible model for the structure and function of the KB-cell receptor for adenovirus is discussed.
An adenovirus-associated protease activity specific for the cleavage of core polypeptide PVII to polypeptide VII was identified and its properties were studied using an in vitro assay system. All temperature-sensitive (ts) mutants examined failed to induce protease activity at 39°. Activity was restored in revertants. The protease activity was completely inhibited by 1 mM tosylamide phenylethylchloromethyl ketone while phenylmethylsulfonylfluoride and tosyl-lysinechloromethyl ketone at 1 mM reduced enzyme activity to 36 and 10% of control, respectively. By contrast ethylenediamine tetraacetic acid had no effect. Optimum enzyme activity was observed at neutral pH. Enzyme activity was stable up to, but not beyond, 45°. Polypeptide PVII was cleaved whether it was in the soluble form, bound form, heat-, or acid-precipitated form. Wild-type young virions contain endogenous protease activity while the virions produced at 39° by tsl-infected cells do not. The PVII contained in these tsl-39 virions, however, may be processed by exogenous WT enzyme after the particles have been frozen-thawed several times. These results suggest that the adenovirus-associated protease is a chymotrypsin-like, nonmetalo, neutral protease.
The uncoating of adenovirus type 2 and a temperature-sensitive mutant, tsl, was studied. HEp-2 cells were infected with 32P- OR 125I-labeled purified virions for various lengths of time, and the nuclear and cytoplasmic fractions were analyzed by sucrose gradient velocity sedimentation and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Within 1 h of infection, virions were converted into three subviral structures: (1) subviral structures in the cytoplasm with a density greater than virions but which qualitatively still contained all virus polypeptides; (ii) corelike structures associated with both the nuclear and cytoplasmic fractions and composed of viral DNA and polypeptides VIa2, V and PVII; and (iii) putative DNA-terminal protein complexes in the nuclei. The kinetic and compartmentalization studies suggested that the DNA-terminal protein complex is the end product of uncoating. The virions which were synthesized by tsl at the nonpermissive temperature and contained the precursor polypeptides PVI and PVII were found to be blocked in uncoating at the corelike stage. This block in uncoating provides the explanation for the lack of infectivity of these virions. A model for the uncoating of adenovirus is proposed.
A technique of destabilization of KB cell plasma membrane by treatment with hypotonic citrate buffer was employed. The technique did not alter the viability or ability of the cells to synthesize macromolecules or produce virus, but did permit the visualization, by the freeze-fracture technique, of plasma membrane modifications occurring in response to adenovirus adsorption. The modifications consisted of a rearrangement of the membrane particles (MPs) on both protoplasmic and external leaflets of the plasma membrane. The rearrangement delineated bare areas, 139 nm in mean diameter, devoid of MPs and protruding outwards. The membrane changes were transient and were only observed when KB cells were maintained with adenovirus particles at 0 °C. The changes disappeared rapidly upon warming to 37 °C, reforming the ‘random pattern’ of MPs, normally visible on the cell plasma membrane. The same type of study was carried out with purified adenovirus capsid components (hexon, penton, penton base and fiber), with smaller virus particles (poliovirus) and with larger ‘artificial’ adenovirus particles made of latex beads coated with adenovirus pentons. The dimensions of the bare regions devoid of MPs appeared to be related to the size of the particles used, suggesting the existence of a ‘recognition pattern’ specific for virus particles.
Using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis of [35S]methionine-labeled adenovirus type 2-infected KB cell extracts, a total of 23 virus-induced polypeptides was detected. This technique was applied to the analysis of the temperature-sensitive mutant, ts 1, which has previously been shown to be defective in a late function. By means of pulse-chase experiments, ts 1 was shown to be defective in the processing of the precursor polypeptide (Pre VII) to the major core protein VII. Two other putative precursor polypeptides, Va (27K) and Vb (24K), were also not processed. Thus, the ts 1 mutation blocked the appearance of six post-translational clevage products, i. e., polypeptides VI, VII, VIII, X, XI, and XII. All of these polypeptides are virion components. Processing was temperature sensitive in a shift-up experiment, whereas it was normal in a shift-down experiment. The kinetics of the temperature-shift experiments suggested that infectious virus could be recovered if enough time is provided for processing to take place. Processing was not inhibited by cycloheximide. The analysis of purified virus particles and empty shells (TCs) revealed the presence of the precursor and putative precursor polypeptides Pre-VII, Va and Vb, instead of their cleavage products, in both types of particles. Based on these results we propose that the ts 1 gene codes for or regulates an endoprotease which is responsible for the completion of the last step in virus maturation, that is, the conversion of "young virions" into mature infectious virions by a series of maturation cleavages.
The early interaction between KB cells and adenovirus was studied by examining the uptake of an extracellular fluorescent macromolecule, FITC (fluorescein isothiocyanate)-labeled dextran (FD). When cells in suspension were incubated with both adenovirus and FD, cell-associated FD increased 2- to 3-fold the value obtained without adenovirus. Under fluorescence microscopy, cells incubated with adenovirus showed bright, whole-cellular fluorescence; whereas, those incubated without adenovirus, or with heat-inactivated virus, showed weaker fluorescence, mainly of the pinocytic vesicles. The increased uptake of the FD by adenovirus was inhibited by treating KB cells with the drugs chloroquine, ammonium chloride and monensin that raise the pH of the acidic compartment. Entry of adenovirus into the KB cell's nucleus also was inhibited by these drugs. The conclusion is that entry of adenovirus into the cell involves its passage of an acidic compartment (probably the endocytic vesicle) and that co-endocytosed macromolecules are released into the cytosol on entry.
Mammalian cells infected with enveloped or naked animal viruses become permeabilized to several proteins. The entry of alpha-sarcin, horseradish peroxidase, and luciferase is greatly increased during the early stages of viral infection. This process is promoted by uv-inactivated SFV, but not by heat-inactivated virions, suggesting that the process does not require viral gene expression. The entry of alpha-sarcin has been monitored both by its effects on protein synthesis and by indirect immunofluorescence. Increased entry of alpha-sarcin and luciferase is clearly observed in animal virus-infected cells by fluorescence microscopy. Chloroquine blocks the coentry of alpha-sarcin with enveloped, but not with naked, viruses. These results have implications to elucidate the mechanisms involved in virus entry.
Restriction endonucleases BamHI, BclI, BglI, BglII, BstEII, EcoRI, HindIII, HpaI, SalI, SmalI, XbalI, and XholI were used to analyze 61 selected strains of adenovirus type 3 (Ad3) isolated from Africa, Asia, Australia, Europe, North America, and South America. It was noted that the use of BamHI, BclI, BglII, HpaI, SalI, and SmaI was sufficient to distinguish 17 genome types; 13 of them were newly identified. All 17 Ad3 genome types could be divided into three genomic clusters. Genome types of Ad3 cluster 1 occurred in Africa, Europe, South America, and North America. Genomic cluster 2 was identified in Africa; genomic cluster 3 was identified in Africa, Asia, Australia, Europe (a few), and North America. This was of interest because 15 identified genome types of Ad7 could also be divided into three genomic clusters. The degree of genetic relatedness between the 17 Ad3 and the 15 Ad7 genome types was analyzed and was expressed in a three-dimensional model.
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.
The adenovirus E1B gene products are required for productive infection of human cells and for complete transformation of rodent cells in cooperation with the E1A gene products. Two major, unrelated polypeptides of 55,000 (55K) and 19,000 (19K) daltons are encoded by the E1B region. The 55K protein is required for efficient DNA replication, late mRNA transport to the cytoplasm and shut-off of cellular mRNA transport in productively infected cells. This protein is required for virus-mediated, but not DNA-mediated, transformation of rodent cells. It appears that the 55K protein does not directly contribute to cell transformation, but influences the oncogenicity of adenoviruses when they are inoculated into newborn hamsters. In contrast, the 19K protein is required for adenovirus induced cellular transformation and oncogenicity and localizes to membranes of the nuclear envelope, cytoplasm and the cell surface in transformed cells. This protein affects the efficiency of virus growth in some, but not all, human cells by regulating the expression of other adenovirus early genes.
Taking advantage of the sedimentation properties of adenovirus particles, adenovirus-infected baby hamster kidney (BHK21) cells were reversibly fixed with cleavable diimidoester dimethyl 3,3'-dithiobispropionimidate (DTBP) at early times of infection (30 min). Cytoskeletal proteins associated with/or in close vicinity to virions were isolated as a complex cross-linked with carrier virus. Four major cellular proteins were thus found to co-purify with adenovirus particles. They were characterized by their coordinates on 2D maps and immunological reactivity. Two of them were identified as alpha-tubulin (58 kD), and vimentin subunits (56 kD). The two other species 68 and 66 kD might correspond to stress proteins. Affinity blotting on gels showed that both alpha-tubulin and vimentin were capable of binding with intact and penton-less adenovirions. Adenovirus components involved in the binding seemed to be mainly core proteins V and VII, and to a lesser extent, hexon. Analysis of neighbor relationships among proteins of the adenovirus-cytoskeletal protein cross-linked complex suggested that some capsid alterations occurred upon/or after entry of the virus into the cell, and that these structural modifications preferentially concerned the vertex components penton and IIIa, and the core protein V.
Evidence is presented that a fraction of vimentin, a component of cytoskeleton recently found to be associated with intracytoplasmic, migrating adenovirus type 2 (Ad2), is processed into smaller polypeptides at early times after infection. The extent of vimentin cleavage appears to depend upon both the multiplicity of infection and the adenovirus serotype. Ad2, Ad5, Ad4, and Ad9 induced similar vimentin cleavage in infected cells, whereas Ad3, Ad7, and Ad12, for which most infecting particles are found sequestered within phagosomes, induced very little, if any, vimentin breakdown. This suggests that vimentin processing is in some way related to the number of virus particles migrating through the cytoplasm. Experiments performed in vitro and in vivo with adenovirus temperature-sensitive mutants H2 ts1 and H2 ts112 and UV-inactivated wild-type Ad2 indicated that vimentin processing is due to a nonvirion, cytoskeleton-associated, proteolytic enzyme activated by adenovirus and sharing characteristics with the protease described by Nelson and Traub (W.J. Nelson and P. Traub, J. Cell Sci. 57:25-49, 1982). The activity of this protease appears to be required for productive infection by adenovirus serotypes 2 and 5 (subgroup C), 4 (subgroup E), and 9 (subgroup D) but not by the oncogenic serotypes 3 and 7 (subgroup B) and 12 (subgroup A).
The first demonstrations of specifically initiated eukaryotic transcription in vitro were published about 10 years ago; both involved transcription by RNA polymerase III. The intervening decade has brought impressive changes to our understanding of eukaryotic transcription in general. This article is devoted to an analysis of current knowledge and thought about transcription by RNA polymerase III. We start with a brief section on this polymerase, which is responsible for synthesis of a variety of small RNAs. There is now an opportunity to identify and sequence genes coding for all the RNA polymerase III subunits, and some of the genes have already been cloned. The newly accessible molecular genetic dissection will tell a lot about phylogeny and may help with understanding function. However, the major focus of this review is on initiation of transcription, on promoters, on transcription factors, and on the formation of promoter complexes. RNA polymerase III promoters are primarily intragenic. Those of the tRNA genes are highly conserved among all the eukaryotes. This conservation reflects the involvement of only a small number of accessory transcription proteins, perhaps likewise relatively highly conserved. On the other hand, the influence of sequence on the rate of initiation of transcription extends into both flanks of polymerase III transcription units. We state a number of the known facts about these extended promoter regions, but this aspect of RNA polymerase III gene promoters has so far received almost no attention at the biochemical level. The current state of knowledge about the best-studied of the initiation factors, TFIIIA, is reviewed. This remarkable protein, with its repeating domain structure, binds along the entire length of an internal control region that covers almost five helical turns of DNA. Progress in understanding the properties of the other transcription factors has been less rapid, somewhat in relation to their lower abundance. Certain functions, including the definition of DNA-binding sites and the formation of stable complexes, can be analyzed without completely purifying them; we deal with these properties at length. In a closing section we consider how the formation of stable complexes with promoters might affect the structure and assembly of chromatin and how chromatin assembly might affect the outcome of competition for transcription factors among genes. That this is a relatively short section, rather than the central theme, of this article reflects the focus of current research; the rules of protein-protein and protein-DNA interactions determining the specific initiation of transcription by RNA polymerase III are largely being worked out in a histoneless frame of reference, following a logic and an analytical repertoire that is shared with studies of prokaryotic gene regulation. However, the assembly of chromatin does cause important and eukaryote-specific consequences to flow from the formation of promoter complexes and we discuss that topic.
Human adenoviruses (e.g., Ad2, Ad5) establish chronic infections in human lymphoid-derived cell lines, including Raji and Jijoye (R.E. Wallace, 1969, Proc. Soc. Exp. Biol. Med. 130, 702-710; N. Faucon, G. Ogier, and Y. Chardonnet, 1982, J. Natl. Cancer Inst. 69, 1215-1220); however, the mechanisms by which chronic infections are established and maintained are not understood. When Raji or MOLT-3 cell cultures were infected with Ad2 at high multiplicity, these cell lines continued to grow exponentially and produced only small amounts of infectious virus. Virus-specific antigens, including the DNA-binding protein and hexon, were expressed in only 5% of the Ad2-inoculated cultures. All Raji and MOLT-3 cells were found to have adenovirus receptors, but the Ad2 virions that adsorbed to most Raji cells were sequestered in caps, suggesting that most cells fail to internalize adsorbed Ad2. Cell synchronization experiments showed a correlation between the proportion of cells that became productively infected and the proportion of cells in mitosis at the time of infection. In contrast, primary blood lymphocytes had few, if any, Ad2 receptors and were not productively infected by Ad2.
A 19-kDa protein, present in low copy number in purified adenovirus type 2, has been characterized. Several criteria were used to establish that this protein is neither a degradation product of the known structural proteins of the virion nor a minor, unusually modified, form of protein VII. This 19-kDa protein, unlike other virion proteins, possesses alkali-resistant phosphoamino acids. Analysis by partial proteolysis indicated that it is related to a 23-kDa phosphoprotein present in H2ts-1 virions assembled in infected cells maintained at 39 degrees C. Affinity labeling with [3H]diisopropyl fluorophosphate showed that the 19-kDa protein contains the active site for a serine protease. We, therefore, conclude that the 19-kDa protein is the active form of the adenovirus-encoded endopeptidase, defined by the H2ts-1 mutation, and is synthesized as a 23-kDa precursor that appears to mature by autocatalysis.
Enteric adenovirus type 40 (Ad40) and Ad41 form the sixth subgenus of human adenoviruses. They are associated with infantile diarrhea but cannot be isolated in conventional cell cultures. The genome of the fastidious enteric Ad41 has been cloned, and the cleavage sites of the genome produced by restriction endonucleases BamHI, EcoRI, HpaI, NruI, PvuI, and SalI have been mapped. To develop useful hybridization methods for direct detection of adenoviruses, a restriction fragment library containing Ad41 DNA, with plasmid pBR322 as vector, has been constructed. Clones have been isolated which contain 8 of 10 possible BamHI fragments of Ad41, inserted into the BamHI cleavage site of the vector. Two of these clones are particularly useful for the detection of adenoviruses. One clone detects members of all human adenovirus subgenera, and the second clone is specific for enteric adenoviruses, in particular Ad41. A conspicuous absence of detectable homology was noted at 1.5 to 3.3 map units of the Ad41 genome in hybridizations against other serotypes of adenoviruses, including the closely related enteric Ad40. This sequence corresponds to the 5' portion of early region Ia.
Highly purified human adenovirus type 2 directly penetrated the plasma membranes of KB cells. The process of membrane penetration resulted in the appearance of large numbers of adenovirions free in the cytoplasm of the infected cells. The virions underwent a morphological change as they penetrated the cell surface. Penetration of the plasma membranes and the accompanying alteration in virion morphology was dependent on a function associated with the intact cells, because neither event occurred when purified virions were added to isolated cell membranes. Inactivation of the adenovirions with heat or antibodies before inoculation of the cells reduced the infectivity of the virus population and prevented the appearance of virions free in the cytoplasm. The inactivation of the virions did not significantly reduce the number of virus particles which were found in cell vacuoles and pinocytotic vesicles.
The chapter discusses the various properties of the adenoviruses, such as the nature of virion and the controlling factors in the productive or abortive infection. The dissection of the virion and its component parts, summarized in this chapter, has provided essential markers for the exploration and correlation of these biological variables, the mapping of phage chromosomes, based on deletions in conditional lethal mutants, required the structural and functional identification of phage precursors and subunits, such that each identifiable moiety of the adenovirion will help in relating biological activity to structural and molecular properties of the viral genomes. For a full understanding of the biology of adenovirus infection, as of other virus-cell systems, it is important to recognize that uniformity and synchrony of response is a deliberately created experimental artifact resulting only from high multiplicity infection of competent cells. This type of response may be absent in natural infections, which does not occur under conditions of low multiplicity infection, and may be fundamentally irrelevant in adenoviral tumorigenesis and abortive infection. For the sake of simplicity, any infection with complete adenovirus particles not leading to production of infectious progeny will be defined as “abortive.” The term “complete” in this context, implies that the same virions can induce productive infection in some suitable indicator cell. Hence, these viruses can be characterized, operationally, as host dependent conditionally lethal, either the permissive host cell supplies some function needed for virus replication that the non permissive cell lacks, or the latter imposes a restriction not present in the permissive one. Both alternatives could, in a purely descriptive way, satisfy the need for working hypotheses to explain the various examples of abortive infection with adenoviruses.
A series of 200 human tumors were cultivated in vitro in an attempt to establish cell lines. Lines were established, with explant and trypsinization techniques, from 13 tumors including carcinomas, sarcomas, melanomas, and brain tumors. All these lines, in culture for over 1 year, exhibited marked refractility, multilayering, and criss crossing and were morphologically distinct from normal contact inhibited human fibroblast or epithelial lines. They also formed colonies on monolayers of normal cells and grew with a high efficiency in soft agar. Preliminary results indicated abnormal chromosomal patterns in all lines tested, and 8 of 9 cell lines formed tumors in antithymocyte serum treated mice. The rate of establishment (approximately 6%) of lines from random neoplastic material demonstrated that cells with properties of transformed cells could be recovered from tumor tissue, but it also emphasized the need for improved methodology in this area.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Comparative experiments with serotypes 1, 5, 7, and 12 revealed genetically controlled differences in penetration. Virions of type 5, belonging to one adenovirus group, possess longer fibers, are adsorbed firmly to the host, and penetrate rapidly in high proportion to the nucleus. Type 7, in another serological group, possesses shorter fibers, becomes less firmly attached, and is preferentially shunted into dense granules (lysosomes), where further penetration is apparently blocked. An electron microscopic analysis revealed that penetration of adenovirus type 1 followed the pattern observed with type 5 while penetration of type 12 was similar to that of adenovirus 7. Some possible explanations are discussed to account for these serotype-controlled differences in the apportionment of the inoculum between various cellular compartments.
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.
Fifteen strains of faecal adenovirus from 3 continents (4 from Europe, 4 from Canada, and 7 from South Africa) were typed as Ad41a by restriction enzyme analysis o the DNA using Sma I. The DNA of the strains was further compared by digestion with 9 other restriction enzymes. All strains had the same cleavage sites for enzymes Bam HI, Bg1 I, Sst I, Pvu II, and Xho I, indicating a close relationship. The Canadian and European strains also had the same cleavage sites for Hind III, Eco RI, Bst EII, and Kpn I, within the limits of resolution of the technique. Only one of the 7 South African strains had a combination of restriction sites identical to the European and Canadian strains, whereas Hind III, Eco RI, Bst EII, and Kpn I digests of the other 6 South African Ad41 DNAs showed the existence of 4 other genome types of Ad41. All 15 Ad41a strains studied were easily distinguished from Ad40 strains using any of the 10 restriction enzymes.
In a prospective 1-year study of acute infantile gastroenteritis, adenoviruses were detected in the stools or by seroconversions, or both, in 56 of 416 (13.5%) ill children. By use of DNA restriction enzyme analysis, enzyme immunoassay, and culture techniques, 33 of 56 (59%) adenovirus specimens were identified as enteric adenoviruses 40 and 41 (Ad40 and Ad41). They were found as the sole recognizable cause of diarrhea in 30 of 416 (7.2%) ill children and in 0 of 200 controls. Three additional ill children had enteric adenoviruses as a part of a dual infection. Evidence for established adenoviruses (Ad1 through Ad39) in gastroenteritis was found in 15 of 416 (3.6%) ill children but also in 3 of 200 (1.5%) controls. Eight adenovirus specimens remained untyped. Seroconversions were demonstrated in 17 of 18 (94%) paired serum samples from patients shedding enteric adenoviruses. The predominant symptom of infections with enteric adenoviruses was diarrhea, with a mean duration of 8.6 days (Ad40) and 12.2 days (Ad41). One-third of the children with Ad41 infections had prolonged symptoms (greater than or equal to 14 days). The frequency of respiratory symptoms was low (21%). The established adenoviruses presented a different clinical picture, characterized by diarrhea of shorter duration, higher fever, and significantly increased occurrence of respiratory symptoms (79%). In conclusion, enteric adenoviruses appear to be an important cause of acute infantile gastroenteritis, second only to rotaviruses in this study.
Enteral adenovirus (EAd) isolates which characteristically fail to propagate in several human cell lines grow readily in 293 cells, an adenovirus type 5 transformed line. EAd infection of 293 cells is associated with high levels of 31S DNA and late viral protein synthesis, the appearance of a progressive adenovirus-like cytopathic effect, and the efficient assembly of progeny virions. The DNA of an EAd strain grown in 293 cells possesses restriction endonuclease profiles similar to those reported for EADs recovered directly from stools. In their ability to replicate efficiently in 293 cells but not in other human cell lines. EAds appear to behave like adenovirus host range mutants.