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C i.p. , C p.p. , and infectivity of HIV-1 virions as a function of harvest time post transfection. Filled circles are for virions produced using 1.0 mg pNL4-3E 2 plasmid and 0.2 mg pcDNA3.1REC. Open circles are for Gag particles produced using 1.0 mg pNL4-3E 2 plasmid only. The dashed line is (C) is fit of the time course to a sum of two-exponential kinetics. Error bars are standard deviations from three independent replicates of the same experiments. doi:10.1371/journal.pone.0067170.g004
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The infectivity of retroviruses such as HIV-1 in plasma or cultured media is less than 0.1% in general, the mechanisms of which are not yet fully understood. One possible explanation among others is the potential presence of large numbers of defective virions in a virus pool, which limits the apparent infectivity of HIV virions. To test this hypoth...
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... provirus together with an envelope plasmid pcDNA3.1REC encoding NL4-3 envelope and collected HIV-1 virions at different times post transfection. For each collection, we determined C i.p. by blue-cell counting and measured C p.p. by p24 ELISA. The infectivity of virions was then calculated as the ratio between these two concentrations. As shown in Fig. 4A, infectious particles can be detected as early as six hours post transfection (filled circles), reaching a peak value around 24 hours and the C i.p. starts to drop off slowly afterwards. In contrast, the concentration of physical particles C p.p. continues to increase until 48 hours and then drops off (Fig. 4B). As a result, the ...
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... these two concentrations. As shown in Fig. 4A, infectious particles can be detected as early as six hours post transfection (filled circles), reaching a peak value around 24 hours and the C i.p. starts to drop off slowly afterwards. In contrast, the concentration of physical particles C p.p. continues to increase until 48 hours and then drops off (Fig. 4B). As a result, the infectivity of virions collected at different times shows a pronounced dependence on harvest time, reaching a maximum at 18 hours and dropping off thereafter (Fig. 4C). The difference in infectivity can be as large as fivefold for virions collected at different time points post transfection. This time dependence has ...
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... C i.p. starts to drop off slowly afterwards. In contrast, the concentration of physical particles C p.p. continues to increase until 48 hours and then drops off (Fig. 4B). As a result, the infectivity of virions collected at different times shows a pronounced dependence on harvest time, reaching a maximum at 18 hours and dropping off thereafter (Fig. 4C). The difference in infectivity can be as large as fivefold for virions collected at different time points post transfection. This time dependence has interesting implications for the production of HIV-1 virions by 293T cells; either the virions produced at early times lose infectivity with time, or more defective virions are produced ...
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... infectivity decay of HIV-1 virions has been reported previously [1], which is attributable to loss of reverse transcriptase activity, biophysical instability of virion particles and possibly gp120 shedding, although the shedding of gp120 was later shown to be insignificant for NL4-3 virus [47]. The decay of HIV-1 virion infectivity in Fig. 4C has a half life of 6.6 hours or greater as determined by fitting the infectivity time course to a two- exponential kinetic model (dashed line). To examine the cause of this infectivity decay, we measured the apparent rate of virion inactivation. These experiments were done by incubating the harvested HIV virions at 37uC in either ...
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... decreases in C p.p. led to an overall increase in infectivity for all of the different virions we have produced. This phenomenon indicates that media change post transfection may remove potential sources of defective virions. Where do these defective virions come from? At six hours post transfection, the level of Gag production is still very low (Fig. 4). The observed decrease in C p.p. due to media change is more than the amount of C p.p. produced at six hours post transfection, suggesting that these defective virions are actually produced more than six hours after transfection. Gag particles that do not carry any envelope proteins may be one potential source of defective virions, ...
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... different mechanisms under current conditions. First, even though the two plasmids were transfected simultaneously during the production of single-cycle virions, some cells may receive only the mutant provirus instead of the two plasmids. We initially produced virions using 1 mg mutant provirus DNA (14.8 kb) and 0.2 mg envelope plasmid (8.6 kb) (Fig. 4). This amount of envelope plasmid was established during our initial optimization for virions collected 48 hours post transfection. Under these conditions, mutant provirus DNA is in , threefold molar excess over the envelope plasmid, which may favor the uptake of mutant provirus only. To test this hypothesis, we transfected 293T cells ...
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... which may give rise to less infectious virions. To test whether uptake of the mutant provirus alone can generate Gag particles under our conditions, we transfected 293T cells with just the mutant provirus DNA and assayed the amount of Gag proteins secreted into culture media using p24 ELISA. The time course of Gag particle production is shown in Fig. 4B as open circles, which displays a time dependent increase that is very similar to the time course of virion production. Infection assays using TZM-bl cells revealed negligible numbers of blue cells (open circles in Fig. 4A) and the resulting infectivity for these particles was five orders of magnitude lower than enveloped virions (Fig. ...
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... amount of Gag proteins secreted into culture media using p24 ELISA. The time course of Gag particle production is shown in Fig. 4B as open circles, which displays a time dependent increase that is very similar to the time course of virion production. Infection assays using TZM-bl cells revealed negligible numbers of blue cells (open circles in Fig. 4A) and the resulting infectivity for these particles was five orders of magnitude lower than enveloped virions (Fig. 4C). Taken together, the above results suggest that there is a mechanism for production of defective particles during the production of single-cycle virions. Cells that receive the mutant provirus only will generate Gag ...
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... shown in Fig. 4B as open circles, which displays a time dependent increase that is very similar to the time course of virion production. Infection assays using TZM-bl cells revealed negligible numbers of blue cells (open circles in Fig. 4A) and the resulting infectivity for these particles was five orders of magnitude lower than enveloped virions (Fig. 4C). Taken together, the above results suggest that there is a mechanism for production of defective particles during the production of single-cycle virions. Cells that receive the mutant provirus only will generate Gag particles without envelope glycoproteins, which reduce the overall infectivity of HIV-1 virions. Media change after ...
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... (Fig. 10C), suggesting that more envelope incorporation into Gag particles increases virion infectivity. Among these observations, we also notice that the infectivity of virions collected 18 hours post transfection with 0.2 mg envelope plasmid is slightly lower than that from 48 hours, which is not readily consistent with our early findings in Fig. 4, where early harvest time favors higher infectivity. This lower infectivity at 18 hours was possibly due to the inclusion of excess vector DNA under this condition, where 3.8 mg vector DNA was included together with 0.2 mg envelope plasmid. This result was confirmed in a side-by-side comparison, where 0.2 mg envelope plasmid was ...
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... These results can be explained by a combination of three different mechanisms: virion spontaneous inactivation, simultaneous production of defective or less infectious particles, and incorporation of envelope glycoprotein into Gag particles. An early harvest time generates virions of higher infectivity. This effect can be as large as fivefold (Fig. 4C). One mechanism behind this phenomenon is the decay of virion infectivity over time as a result of biophysical instability (Fig. 5), which competes with the production of infectious virions. Virions harvested at later times post transfection contain more defective virions, which results in an apparent lower infectivity. However, this is ...
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... particles are also produced by the cells together with infectious virions, which lowers the infectivity of a virus pool. When 293T cells are transfected with a mixture of mutant provirus and envelope plasmids, cells that carry the mutant provirus only will produce defective particles, the concentration of which increases with incubation time (Fig. 4B). Cells that carry both plasmids but have low envelope plasmid will produce less infectious particles (Fig. 10A). The presence of these defective or less infectious particles in a virus pool decreases the overall infectivity. More envelope DNA during transfection favors cells that carry both plasmids, and the increased expression of ...
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... The infectivity of retroviruses, including HIV-1, either in plasma or cultured media, has been reported to be less than 0.1%, arguing potentially large numbers of defective virions in a virus pool and limiting the infectivity of HIV virions [97]. Nonetheless, Josefsson et al. demonstrated that in peripheral blood CD4 + T cells from patients, the majority of infected cells contain only one copy of HIV-1 DNA as compared to the high proviral HIV content present in tissues and co-cultures which may correlate to transmission by cell-free particles [98,99]. ...
HIV mainly targets CD4+ T cells, from which Th17 cells represent a major cell type, permissive, and are capable of supporting intracellular replication at mucosal sites. Th17 cells possess well-described dual roles, while being central to maintaining gut integrity, these may induce inflammation and contribute to autoimmune disorders; however, Th17 cells’ antiviral function in HIV infection is not completely understood. Th17 cells are star players to HIV-1 pathogenesis and a potential target to prevent or decrease HIV transmission. HIV-1 can be spread among permissive cells via direct cell-to-cell and/or cell-free infection. The debate on which mode of transmission is more efficient is still ongoing without a concrete conclusion yet. Most assessments of virus transmission analyzing either cell-to-cell or cell-free modes use in vitro systems; however, the actual interactions and conditions in vivo are not fully understood. The fact that infected breast milk, semen, and vaginal secretions contain a mix of both cell-free viral particles and infected cells presents an argument for the probability of HIV taking advantage of both modes of transmission to spread. Here, we review important insights and recent findings about the role of Th17 cells during HIV pathogenesis in mucosal surfaces, and the mechanisms of HIV-1 infection spread among T cells in tissues.
... The transfection reagent mixture was incubated at room temperature for 15 min before dropwise addition to the culture media, as we did previously. 40 At 6 h post transfection, the culture media together with the transfection reagents was replaced with fresh complete media, and the incubation was continued at 37°C with 5% CO 2 . At 48 h post transfection, the entire culture media containing single-cycle HIV-1 viruses was collected and filtered through a 0.45 μm syringe filter (Millex-HV poly-(vinylidene difluoride) (PVDF), Millipore). ...
... The concentration of virion particles was quantitated using an HIV-1 p24 ELISA kit (CAT#XB-1000, XpressBio) as we described previously. 40 Virion Neutralization Assay. Virion neutralization assay follows the protocols we established previously but with important modifications. ...
... Virion neutralization assay follows the protocols we established previously but with important modifications. 40 HIV-1 virions pseudotyped with SARS-CoV-2 envelope containing 18 ng of HIV-1 p24 were incubated with various mouse sera at 20°C for 1 h and then diluted with complete media by 25-fold in volume for the sera to initiate infection of Huh-7.5 cells at 37°C for 2 h. At the end of 2 h, fresh media was added to each well in a 12-well plate, and the incubation was continued at 37°C with 5% CO 2 . ...
Understanding immune responses toward viral infection will be useful for potential therapeutic intervention and offer insights into the design of prophylactic vaccines. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. To understand the complex immune responses toward SARS-CoV-2 infection, here we developed a method to express and purify the recombinant and engineered viral receptor-binding domain (RBD) to more than 95% purity. We could encapsulate RNA molecules into the interior of a virion-sized liposome. We conjugated the purified RBD proteins onto the surface of the liposome in an orientation-specific manner with defined spatial densities. Both the encapsulation of RNAs and the chemical conjugation of the RBD protein on liposome surfaces were stable under physiologically relevant conditions. In contrast to soluble RBD proteins, a single injection of RBD-conjugated liposomes alone, in the absence of any other adjuvants, elicited RBD-specific B cell responses in BALB/c mice, and the resulting animal sera could potently neutralize HIV-1 pseudovirions that displayed the SARS-CoV-2 spike proteins. These results validate these supramolecular structures as a novel and effective tool to mimic the structure of enveloped viruses, the use of which will allow systematic dissection of the complex B cell responses to SARS-CoV-2 infection.
... The Luciferase reporter based TZM-bl cell line was used to measure HIV-1 infectivity (Kim et al. [38]) after exposure with either HIV-positive U1 Exo/J-Lat(9.2) Exo or HIVnegative U937 Exo/Jurkat Exo. Briefly, TZM-bl cells (5 × 10 4 cells/well) were cultured in a 48-well plate one day before the experiment. ...
Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders. To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of primary human brain microvascular endothelial cells (HBMVECs) by inducing mitochondrial hyperfusion, and reducing the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS). The quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. It was further supported by the detection of exosomal markers, and the absence of large EV-related protein in the isolated EVs. The exosomes were readily taken up by primary HBMVECs. HIV-exosomes induce cellular and mitochondrial superoxide production but reduce mitochondrial membrane potential in HBMVECs. HIV-exosomes increase mitochondrial hyperfusion, possibly due to loss of phosphorylated dynamin-related protein 1 (p-DRP1). HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein reduce the expression of p-DRP1 and p-eNOS, and accelerate brain endothelial dysfunction. Finally, exosomes isolated from HIV-1 infected primary human peripheral blood mononuclear cells (hPBMCs) produce more exosomes than uninfected controls and reduce both p-DRP1 and p-eNOS expressions in primary HBMVECs. Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium.
... [73][74][75][76] However, there are few demonstrations on plasmonic trapping of lipid vesicles, which are essential to study biological mechanisms. 77,78 Several challenges exist besides the aforementioned issue of biosafety, such as the difference in the refractive indices between biological objects and the surrounding medium, the intrinsically low dipole moments due to spherical morphology, and the relatively small size of lipid vesicles. 74,79 Recently, Zheng's group reported a simple optical setup to trap lipid vesicles through SP induced localized heating and optical scattering forces. ...
Plasmonic tweezers are an emerging research topic because of their breakthrough to the conventional diffraction limit and precise manipulation at nanoscale. Notably, their compatibility with analytical techniques (e.g. fluorescence, surface-enhanced Raman scattering (SERS), and laser desorption/ionization mass spectrometry (LDI MS)) opens up opportunities in optical manipulation and biomedical applications. Herein, we firstly introduce the structures and trapping forces, followed by a summary of the properties to better characterize plasmonic tweezers. The optical trapping of biosamples by plasmonic tweezers are then reviewed, including microorganisms and biomolecules. Finally, we highlight the integration of plasmonic tweezers with analytical techniques towards the bioanalytical applications. We conclude with perspectives on the future directions for this topic. We foresee the upcoming era of biological detection by plasmonic tweezing in both academy and industry, which calls for the interest and effort of scientists from diverse fields.
... Luciferase reporter assays were carried out to measure the viral infectivity using the TZM-bl cell line [48]. Briefly, cells (2 × 10 4 /well) were cultured in a 24-well plate one day before the experiment. ...
... We observed that short-term exposure (2 h) to low-dose CyO2 (0.1-1.5 µM) markedly decreased HIV p24 levels in the viral pellets, which indicated the disruption of intact viral particles by CyO2 ( Figure 7B). To further confirm that CyO2-induced viral disruption can decrease infectivity, a highly quantitative HIV-infectivity assay was employed by using the TZM-bl cell line [48]. Percent change in relative luciferase units (RLU) clearly showed a 4-fold increase in cells exposed to HTLV-IIIB virus. ...
... cell line [48]. Percent change in relative luciferase units (RLU) clearly showed a 4-fold increase in cells exposed to HTLV-IIIB virus. ...
Background: Novel strategies to increase the efficacy of antiretroviral (ARV) drugs will be of crucial importance. We hypothesize that membranes of HIV-1-infected cells and enveloped HIV-1 particles may be preferentially targeted by the phytopeptide, cycloviolacin O2 (CyO2) to significantly enhance ARV efficacy. Methods: Physiologically safe concentrations of CyO2 were determined via red blood cell (RBC) hemolysis. SYTOX-green dye-uptake and radiolabeled saquinavir (3H-SQV) uptake assays were used to measure pore-formation and drug uptake, respectively. ELISA, reporter assays and ultracentrifugation were conducted to analyze the antiviral efficacy of HIV-1 protease and fusion inhibitors alone and co-exposed to CyO2. Results: CyO2 concentrations below 0.5 μM did not show substantial hemolytic activity, yet these concentrations enabled rapid pore-formation in HIV-infected T-cells and monocytes and increased drug uptake. ELISA for HIV-1 p24 indicated that CyO2 enhances the antiviral efficacy of both SQV and nelfinavir. CyO2 (< 0.5 μM) alone decreases HIV-1 p24 production, but it did not affect the transcription regulatory function of the HIV-1 long terminal repeat (LTR). Ultracentrifugation studies clearly showed that CyO2 exposure disrupted viral integrity and decreased the p24 content of viral particles. Furthermore, direct HIV-1 inactivation by CyO2 enhanced the efficacy of enfuvirtide. Conclusions: The membrane-active properties of CyO2 may help suppress viral load and augment antiretroviral drug efficacy.
... To this end, we analyzed the effect of TfR overexpression and incorporation into the viral membrane by transfecting different amounts of TfR plasmid into 293T virus-producing cells along with fixed amounts of viral plasmids. We performed infectivity assays using TZM-bl cells, as described [21], To assess the effect of TfR expression and the presence of exogenous biotin on virus production in 293T cells, we performed an enzyme-linked immunosorbent assay (ELISA) to detect HIV-1 p24 capsid protein. Figure 3A shows the concentration of virion particles as a function of TfR plasmid (pTfR) input during transfection for two strains of HIV-1 with two different Env. ...
... The procedures to produce biotinylated HIV-1 virions are modified based on our procedures to produce HIV-1 virions as published previously [21]. Briefly, pTfR-AP-IRES-BirA-ER (hereafter pTfR, for simplicity) [32] was subcloned into the pcDNA3.1(+) ...
... The following plasmid amounts were added to each 35 mm well. For EGFP-Vpr virions, 293T cells were transfected with 1 μg pNL4-3R-E-, 1 μg envelope plasmid encoding either a fully functional NL4-3 or BG505 envelope glycoprotein gp120/ gp41 as indicated in the text, 0.3 μg pEGFP-Vpr [21] and varying amounts of pTfR with TransIT LT-1 transfection reagent (Mirus Bio, Madison, WI). For iGFP virions, 293T cells were transfected with 1 μg pNL4-3-iGFP2 and different envelope plasmids. ...
Direct optical trapping of single viral particles allows characterization of individual particles in suspension with single-molecule sensitivity. Alternative to direct optical trapping of particles, individual particles may be tethered specifically in suspension for manipulation by optical tweezers indirectly, which could be useful for studies of virus-cell interactions. One specific example is the interactions between cell surface receptors and the envelope glycoproteins (Env) on the surface of human immunodeficiency virus type 1 (HIV-1). Env binds to cellular receptors and undergoes a series of conformational changes, culminating in fusion of the viral and cellular membranes that mediates viral entry into cells. In addition to being required for cellular infection, Env is also the sole target for neutralizing antibodies. Thus, significant research has focused on elucidating the structure of Env and the mechanism of HIV-1 entry. However, current methods are unable to resolve the dynamics and stoichiometry of Env binding to cellular receptors during the entry process. Fluorescence and electron microscopy have visualized Env clusters in the viral membrane, but the extent to which these clusters actually bind to cellular receptors, and the mechanism of cluster formation, remain unclear. We describe the development of an optical tweezers technique that can potentially address these questions by delivering a single HIV-1 virion to a live cell with minimal perturbation to the system. Our method can be used to quantitatively probe the physical interactions between Env and cellular receptors in their native environment, which may reveal critical parameters in HIV-1 entry. Furthermore, our method can be used to investigate other protein-protein interactions in the context of live cells, such as the recognition of particulate antigens by B cells, thus offering insight into fundamental features of protein-mediated receptor activation.
... Presumably, by binding to P. gingivalis vesicles, HIV-1 particles, even at non-infectious doses, can now enter permissive cells using the vesicles as a vehicle. It was previously estimated that only 0.01-1% of HIV-1 particles in a culture sample are infectious [38][39][40][41][42][43][44][45] . In other words, the majority of virions cannot infect HIV-1 susceptible cells due to defects in their viral proteins or genes. ...
The association between mucosal microbiota and HIV-1 infection has garnered great attention in the field of HIV-1 research. Previously, we reported a receptor-independent HIV-1 entry into epithelial cells mediated by a Gram-negative invasive bacterium, Porphyromonas gingivalis. Here, we present evidence showing that P. gingivalis outer membrane vesicles (OMVs) promote mucosal transmission of HIV-1. We demonstrated, using the Dynabeads technology, a specific interaction between HIV-1 and P. gingivalis OMVs which led to an OMV-dependent viral entry into oral epithelial cells. HIV-1 was detected in human oral keratinocytes (HOKs) after a 20 minute exposure to the HIV-vesicle complexes. After entry, most of the complexes appeared to dissociate, HIV-1 was reverse-transcribed, and viral DNA was integrated into the genome of HOKs. Meanwhile, some of the complexes exited the original host and re-entered neighboring HOKs and permissive cells of HIV-1. Moreover, P. gingivalis vesicles enhanced HIV-1 infection of MT4 cells at low infecting doses that are not able to establish an efficient infection alone. These findings suggest that invasive bacteria and their OMVs with ability to interact with HIV-1 may serve as a vehicle to translocate HIV through the mucosa, establish mucosal transmission of HIV-1, and enhance HIV-1 infectivity.
... vector for expression in 293T cells using EcoRI and NotI restriction sites. HIV-1 virions were produced as described (Kim et al., 2013) For iGFP virions, 293T cells were transfected with 1 ug pNL4-3-iGFP2 and different envelope plasmids. Before transfection, medium was supplemented with 100 uM biotin, which was previously shown to be saturating for BirA in 293T cells (Nesbeth et al., 2006). ...
... Before transfection, medium was supplemented with 100 uM biotin, which was previously shown to be saturating for BirA in 293T cells (Nesbeth et al., 2006). HIV-1 concentration was assayed with a p24 enzyme-linked immunosorbent assay (ELISA) as previously described (Kim et al., 2013) using the HIV-1 p24 Antigen Capture Kit (Advanced Bioscience Laboratories, Rockville, MD), following the manufacturers instructions. p24 concentration was converted to virion concentration via the assumption of 10 million virions per ng p24 (Kim et al., 2013). ...
... HIV-1 concentration was assayed with a p24 enzyme-linked immunosorbent assay (ELISA) as previously described (Kim et al., 2013) using the HIV-1 p24 Antigen Capture Kit (Advanced Bioscience Laboratories, Rockville, MD), following the manufacturers instructions. p24 concentration was converted to virion concentration via the assumption of 10 million virions per ng p24 (Kim et al., 2013). The concentration of infectious virions (titer) was measured using the TZM-bl indicator cell line, as described (Kim et al., 2013). ...
Kinesins are cytoskeletal motor proteins that transport cargoes along microtubules in eukaryotic cells. Motors in the kinesin superfamily share a highly conserved structure containing two motor domains that dimerize through a coiled-coil stalk. The canonical view is that dimerization is required for kinesin's processive motility and force generation, as the two motor domains of a dimer step along the microtubule lattice in a tightly coordinated manner. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their monomeric forms. Surprisingly, we find that monomeric motors across different kinesin families are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are significantly less efficient at dispersion of the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate up to 11 pN of force in an optical trap. The ability of KIF3B to work in teams enabled us to test the impact of monomer length on collective cargo transport and force generation. We demonstrate that increasing the motor-to-cargo distance results in a decreased efficiency of cellular cargo transport and a decreased speed and force output in vitro. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization is thus critical for cellular transport events that require an ability to generate or withstand high forces. Our findings lend insight into the minimal requirements and mechanical modulators of collective kinesin cargo transport.
... HEK 293T/17 cells (ATCC, Manassas, VA) were cultured at 37°C with 5% CO 2 in DMEM supplemented with 10% FBS. The procedures to produce HIV-1 virions have been described in detail elsewhere [27]. Briefly, the EGFP-labeled HIV-1 virions were produced by transient transfection of 293T cells with two plasmids using Mirus TransIT LT-1 transfection regent. ...
Although Ashkin and Dziedzic first demonstrated optical trapping of individual tobacco mosaic viruses in suspension as early as 1987, this pioneering work has not been followed up only until recently. Using human immunodeficiency virus type 1 (HIV-1) as a model virus, we have recently demonstrated that a single HIV-1 virion can be stabled trapped, manipulated and measured in physiological media with high precision. The capability to optically trap a single virion in suspension not only allows us to determine, for the first time, the refractive index of a single virus with high precision, but also quantitate the heterogeneity among individual virions with single-molecule resolution, the results of which shed light on the molecular mechanisms of virion infectivity. Here we report the further development of a set of microscopic techniques to physically deliver a single HIV-1 virion to a single host cell in solution. Combined with simultaneous epifluorescence imaging, the attachment and dissociation events of individual manipulated virions on host cell surface can be measured and the results help us understand the role of diffusion in mediating viral attachment to host cells. The establishment of these techniques opens up new ways for investigation of a wide range of virion-cell interactions, and should be applicable for study of B cell interactions with particulate antigens such as viruses.
... In this paper, we introduce a quantitative approach to this problem. In our approach, we use a provirus clone that is env Ϫ together with a separate wild-type env plasmid (pEnv) to generate HIV-1 virions (9). It is essential that the provirus is env Ϫ so that we can titrate pEnv from low to high quantities (8) to generate a set of virions that are expected to carry varied densities of Env on their surface, limited by the pEnv inputs (10). ...
... Production of HIV-1 Virions-Single-cycle HIV-1 virions were generated and assayed as described recently (9). Briefly, 293T cells were transfected with 1.0 g of pNL4 -3R Ϫ E Ϫ plasmid, various amounts of pEnv (NL4-3 envelope expression plasmid, the same as pcDNA3.1REC in Ref. 9) and 0.3 g of pEGFP-Vpr by the use of Mirus LT-1 transfection reagents in a 2-ml culture volume in a 35-mm dish. ...
... Production of HIV-1 Virions-Single-cycle HIV-1 virions were generated and assayed as described recently (9). Briefly, 293T cells were transfected with 1.0 g of pNL4 -3R Ϫ E Ϫ plasmid, various amounts of pEnv (NL4-3 envelope expression plasmid, the same as pcDNA3.1REC in Ref. 9) and 0.3 g of pEGFP-Vpr by the use of Mirus LT-1 transfection reagents in a 2-ml culture volume in a 35-mm dish. Culture medium was changed 6 h post-transfection and virions were harvested 24 h post-transfection (9). ...
The envelope glycoprotein (Env) gp120/gp41 is required for HIV-1 infection of host cells. Although in general it has been perceived that more Env gives rise to higher infectivity, the precise quantitative dependence of HIV-1 virion infectivity on Env density has remained unknown. Here we have developed a method to examine this dependence. This method involves (1) production of a set of single-cycle HIV-1 virions with varied density of Env on their surface; (2) site-specific labeling of Env-specific antibody Fab with a fluorophore at high efficiency, and (3) optical trapping virometry to measure the number of gp120 molecules on individual HIV-1 virions. The resulting gp120 density per virion is then correlated with the infectivity of the virions measured in cell culture. In the presence of DEAE-dextran, the polycation known to enhance HIV-1 infectivity in cell culture, virion infectivity follows gp120 density as a sigmoidal dependence and reaches an apparent plateau. This quantitative dependence can be described by a Hill equation, with Hill coefficient of 2.4 ± 0.6. In contrast, in the absence of DEAE-dextran, virion infectivity increases monotonically with gp120 density and no saturation is observed under the experimental conditions. These results provide the first quantitative evidence that Env trimers cooperate on the virion surface to mediate productive infection by HIV-1. Moreover, as a result of the low number of Env trimers on individual virions, the number of additional Env trimers per virion that is required for the optimal infectivity will depend on the inclusion of facilitating agents during infection.
