Inactivation of non-enveloped virus by 1,5 iodonaphthyl azide.

BACKGROUND: A photoactive hydrophobic agent 1,5-iodonaphthyl-azide (INA), has been previously shown to completely inactivate the enveloped viruses. INA sequesters into the lipid bilayer of the virus envelope and upon UV-irradiation bind to the hydrophobic domains of the envelope glycoproteins. In our earlier study, we have shown that the Venezuelan equine encephalitis virus (VEEV) genomic RNA was also inactivated during the inactivation of the virus with INA. FINDINGS: In the present study, we evaluated if the RNA inactivation property of INA can be used to inactivate non-enveloped RNA viruses. Encephalomyocarditis virus (EMCV) was used as a model non-enveloped virus. Treatment with INA followed by UV-irradiation resulted in complete inactivation of EMCV. RNA isolated from INA-inactivated EMCV was non-infectious and INA was found to be associated with the viral RNA genome. INA-inactivated EMCV induced robust total antibody response. However binding capacity of INA-inactivated EMCV to neutralizing antibody was inhibited. CONCLUSION: This is the first study to show that INA can completely inactivate non-enveloped virus. Our results suggest that the amino acid composition of the neutralizing epitope may interfere with the protective antibody response generated by the INA-inactivated non-enveloped virus.

Mycoplasma gallisepticum inactivated by targeting the hydrophobic domain of the membrane preserves surface lipoproteins and induces a strong immune response.

An innovative approach for inactivation of Mycoplasma gallisepticum using the hydrophobic photoinduced alkylating probe 1, 5-iodonaphthylazide (INA) is described. Treatment of washed M. gallisepticum mid-exponential culture (0.2 mg cell protein /mL) with INA followed by irradiation with far-ultraviolet light (310-380 nm) completely abolished viability. Transmission electron microscopy showed that the majority of the inactivated M. gallisepticum were comparable in size to intact cells, but that part of the INA-treated M. gallisepticum preparation also contained low density cells and membrane vesicles. Confocal microscopy revealed that untreated M. gallisepticum cells were internalized by chicken red blood cells (c-RBCs), whereas the INA-inactivated cells remained attached to the outer surface of the c-RBCs. INA treatment of M. gallisepticum resulted in a complete inactivation of F0F1 -ATPase and of the L-arginine uptake system, but the cytoplasmatic soluble NADH2 dehydrogenase was only partially affected. Western blot analysis of the lipoprotein fraction showed that the INA-treated M. gallisepticum retained their lipoproteins. Following subcutaneous injection of M. gallisepticum INA-bacterin, 100% and 68.8% of chickens were positive by the rapid serum agglutination test and enzyme-linked immunosorbent assay respectively, 2 weeks post-injection. These data suggest that the photoinducible alkylating agent INA inactivates M. gallisepticum but preserves its surface lipoproteins and thus has the potential to be used as a general approach for the inactivation of mycoplasmas for vaccine development.

Dengue virus photo-inactivated in presence of 1,5-iodonaphthylazide (INA) or AMT, a psoralen compound (4'-aminomethyl-trioxsalen) is highly immunogenic in mice.

Two novel methods of dengue virus inactivation using iodonaphthyl azide (INA) and aminomethyl trioxsalen (AMT) were compared with traditional virus inactivation by formaldehyde. The AMT inactivated dengue-2 virus retained its binding to a panel of 5 monoclonal antibodies specific for dengue-2 envelope protein, whereas inactivation by formaldehyde and INA led to 30-50% decrease in binding. All three inactivated viruses elicited high level virus neutralizing antibodies in vaccinated mice. However, only mice vaccinated with AMT inactivated virus mounted T cell responses similar to live, uninactivated virus.

Ectopic ATP synthase facilitates transfer of HIV-1 from antigen-presenting cells to CD4(+) target cells.

Antigen-presenting cells (APCs) act as vehicles that transfer HIV to their target CD4(+) cells through an intercellular junction, termed the virologic synapse. The molecules that are involved in this process remain largely unidentified. In this study, we used photoaffinity labeling and a proteomic approach to identify new proteins that facilitate HIV-1 transfer. We identified ectopic mitochondrial ATP synthase as a factor that mediates HIV-1 transfer between APCs and CD4(+) target cells. Monoclonal antibodies against the ß-subunit of ATP synthase inhibited APC-mediated transfer of multiple strains HIV-1 to CD4(+) target cells. Likewise, the specific inhibitors of ATPase, citreoviridin and IF1, completely blocked APC-mediated transfer of HIV-1 at the APC-target cell interaction step. Confocal fluorescent microscopy showed localization of extracellular ATP synthase at junctions between APC and CD4(+) target cells. We conclude that ectopic ATP synthase could be an accessible molecular target for inhibiting HIV-1 proliferation in vivo.

Orthogonal inactivation of influenza and the creation of detergent resistant viral aggregates: towards a novel vaccine strategy.

BACKGROUND: It has been previously shown that enveloped viruses can be inactivated using aryl azides, such as 1-iodo-5-azidonaphthalene (INA), plus UVA irradiation with preservation of surface epitopes in the inactivated virus preparations. Prolonged UVA irradiation in the presence of INA results in ROS-species formation, which in turn results in detergent resistant viral protein fractions. RESULTS: Herein, we characterize the applicability of this technique to inactivate influenza. It is shown that influenza virus + INA (100 micromolar) + UVA irradiation for 30 minutes results in a significant (p < 0.05) increase in pelletablehemagglutinin after Triton X-100 treatment followed by ultracentrifugation. Additionally, characterization of the virus suspension by immunogold labeling in cryo-EM, and viral pellet characterization via immunoprecipitation with a neutralizing antibody, shows preservation of neutralization epitopes after this treatment. CONCLUSION: These orthogonally inactivated viral preparations with detergent resistant fractions are being explored as a novel route for safe, effective inactivated vaccines generated from a variety of enveloped viruses.

Effects of UVA irradiation, aryl azides, and reactive oxygen species on the orthogonal inactivation of the human immunodeficiency virus (HIV-1).

Previously we reported that hydrophobic aryl azides partition into hydrophobic regions of the viral membrane of enveloped viruses and inactivate the virus upon UVA irradiation for 2 min. Prolonged irradiation (15 min) resulted in viral protein aggregation as visualized via Western blot analysis, due to reactive oxygen species (ROS) formation, with preservation of the surface antigenic epitopes. Herein, we demonstrate that these aggregates show detergent resistance and that this property may be useful towards the creation of a novel orthogonal virus inactivation strategy for use in preparing experimental vaccines. When ROS-modified HIV virus preparations were treated with 1% Triton X-100, there was an increase in the percent of viral proteins (gp41, p24) in the viral pellet after ultracentrifugation through sucrose. Transmission electron microscopy (TEM) of these detergent-resistant pellets shows some recognizable virus fragments, and immunoprecipitation studies of the gp41 aggregates suggest the aggregation is covalent in nature, involving short-range interactions.

Characterization of the effects of aryl-azido compounds and UVA irradiation on the viral proteins and infectivity of human immunodeficiency virus type 1.

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4'-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non-azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.

Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

BACKGROUND: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated. RESULTS: INA treatment of various cell lines followed by irradiation with UV light (310-380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor. CONCLUSION: INA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.

HIV-1 envelope glycoprotein-mediated fusion and pathogenesis: implications for therapy and vaccine development.

Our overall goal is to understand how viral envelope proteins mediate membrane fusion and pathogenesis. Membrane fusion is a crucial step in the delivery of the viral genome into the cell resulting in infection. On the other hand, fusion activity of viral envelope glycoproteins expressed in infected cells may cause the demise of uninfected bystander cells by apoptosis. Our general approach is to kinetically resolve steps in the pathway of viral envelope glycoprotein-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. Since HIV fusion involves a complex cascade of interactions of the envelope glycoprotein with two receptors, membrane organization plays an important role and interfering with it may modulate entry. To study this phenomenon, we have either examined cell lines with differential expression of sphingolipids (such as GM3), or altered membrane organization by modifying levels of cholesterol, ceramides, or glycosphingolipids. We show that the localized plasma membrane lipid microenvironment (and not the specific membrane lipids) in the vicinity of CD4 controls receptor mobility and HIV-1 fusion. The complex cascade of conformational changes that must occur to allow virus entry is also a very important target for therapy and vaccine development. We have recently designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to promote permanent attachment. Using a temperature-arrested state in vitro assay we show evidence for the trapping of a pre-six-helix bundle fusion intermediate by a covalent reaction with the inhibitory reactive peptide. Also, using photo-reactive hydrophobic probes we have found ways to inactivate viral envelope glycoproteins while leaving their overall structures intact. Finally, in order to study the envelope glycoprotein effects on pathogenesis, we have used an in vitro model of co-culture of envelope-expressing cells as effectors and CD4+ T cells as targets. We delineated that apoptosis mediated by envelope glycoprotein in bystander cells correlates with transmembrane subunit (gp41)-induced hemifusion. The apoptotic pathway initiated by this interaction involves caspase-3-dependent mitochondrial depolarization and reactive oxygen species production, which depends on the phenotype of the envelope glycoprotein associated with the virus. Taken as a whole, our studies have many different important implications for antiviral therapies and vaccine development.

Hydrophobic inactivation of influenza viruses confers preservation of viral structure with enhanced immunogenicity.

The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to the influenza virus. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound 1,5-iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose-dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH-dependent lipid mixing, but pH-dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza virus induced potent anti-influenza virus serum antibody and T-cell responses, similar to live virus immunization, and protected against heterosubtypic challenge. INA treatment of influenza A virus produced a virus that is noninfectious, intact, and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and hemagglutinin. When used as a vaccine given intranasally (i.n.), INA-inactivated influenza virus induced immune responses similar to live virus infection.

Photoinduced reactivity of the HIV-1 envelope glycoprotein with a membrane-embedded probe reveals insertion of portions of the HIV-1 Gp41 cytoplasmic tail into the viral membrane.

The interactions of HIV-1 Env (gp120-gp41) with CD4 and coreceptors trigger a barrage of conformational changes in Env that drive the membrane fusion process. Various regions of gp41 have profound effects on HIV entry and budding. However, the precise interactions between gp41 and the membrane have not been elucidated. To examine portions of membrane proteins that are embedded in membrane lipids, we have studied photoinduced chemical reactions in membranes using the lipid bilayer specific probe iodonaphthyl azide (INA). Here we show that in addition to the transmembrane anchor, amphipatic sequences in the cytoplasmic tail (CT) of HIV-1 gp41 are labeled by INA. INA labeling of the HIV-1 gp41 CT was similar whether wild-type or a mutant HIV-1 was used with uncleaved p55 Gag, which does not allow entry. These results shed light on the disposition of the HIV-1 gp41 CT with respect to the membrane. Moreover, our data have general implications for topology of membrane proteins and their in situ interactions with the lipid bilayer.

Ebola virus inactivation with preservation of antigenic and structural integrity by a photoinducible alkylating agent.

Current methods for inactivating filoviruses are limited to high doses of irradiation or formalin treatment, which may cause structural perturbations that are reflected by poor immunogenicity. In this report, we describe a novel inactivation technique for Zaire Ebola virus (ZEBOV) that uses the photoinduced alkylating probe 1,5-iodonaphthylazide (INA). INA is incorporated into lipid bilayers and, when activated by ultraviolet irradiation, alkylates the proteins therein. INA treatment of ZEBOV resulted in the complete loss of infectivity in cells. Results of electron microscopy and virus-capture assays suggested the preservation of conformational surface epitopes. Challenge with 50,000 pfu of INA-inactivated, mouse-adapted ZEBOV did not cause disease or death in mice. A single vaccination with INA-inactivated ZEBOV (equivalent to 5 x 10(4) pfu) protected mice against lethal challenge with 1000 pfu of ZEBOV. INA-inactivated virus induced a protective response in 100% of mice when administered 3 days before challenge. Thus, INA may have significant potential for the development of vaccines and immunotherapeutics for filoviruses and other enveloped viruses.

Complete inactivation of Venezuelan equine encephalitis virus by 1,5-iodonaphthylazide.

Hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA) partitions into biological membranes and accumulates selectively into the hydrophobic domain of the lipid bilayer. Upon irradiation with far UV light, INA binds selectively to transmembrane proteins in the viral envelope and renders them inactive. Such inactivation does not alter the ectodomains of the membrane proteins thus preserving the structural and conformational integrity of immunogens on the surface of the virus. In this study, we have used INA to inactivate Venezuelan equine encephalitis virus (VEEV). Treatment of VEEV with INA followed by irradiation with UV light resulted in complete inactivation of the virus. Immuno-fluorescence for VEEV and virus titration showed no virus replication in-vitro. Complete loss of infectivity was also achieved in mice infected with INA treated plus irradiated preparations of VEEV. No change in the structural integrity of VEEV particles were observed after treatment with INA plus irradiation as assessed by electron microscopy. This data suggest that such inactivation strategies can be used for developing vaccine candidates for VEEV and other enveloped viruses.

Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide.

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[(125)I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [(125)I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope.

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.

The HIV Env-mediated fusion reaction.

The current general model of HIV viral entry involves the binding of the trimeric viral envelope glycoprotein gp120/gp41 to cell surface receptor CD4 and chemokine co-receptor CXCR4 or CCR5, which triggers conformational changes in the envelope proteins. Gp120 then dissociates from gp41, allowing for the fusion peptide to be inserted into the target membrane and the pre-hairpin configuration of the ectodomain to form. The C-terminal heptad repeat region and the leucine/isoleucine zipper region then form the thermostable six-helix coiled-coil, which drives the membrane merger and eventual fusion. This model needs updating, as there has been a wealth of data produced in the last few years concerning HIV entry, including target cell dependencies, fusion kinetic data, and conformational intermediates. A more complete model must include the involvement of membrane microdomains, actin polymerization, glycosphingolipids, and possibly CD4 and chemokine signaling in entry. In addition, kinetic experiments involving the addition of fusion inhibitors have revealed some of the rate-limiting steps in this process, adding a temporal component to the model. A review of these data that may require an updated version of the original model is presented here.

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1.

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.

Fluorescent lipid probes in the study of viral membrane fusion.

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.

Quantitative measurement of fusion of HIV-1 and SIV with cultured cells using photosensitized labeling.

The fusion of HIV and SIV with biological membranes was studied by photosensitized activation of a hydrophobic probe, [(125)I]iodonaphthylazide ([(125)I]INA), by a fluorescent lipid which is situated in the target membrane. Photosensitized labeling of viral envelope-resident proteins occurs only upon their insertion into target membranes. Photosensitized labeling as a result of HIV-1 Env-mediated cell fusion showed the same kinetics as aqueous dye transfer. We have for the first time measured kinetics of HIV and SIV virus-cell fusion. HIV-1(MN) virions were about 10x less fusion active than SIVmne virions. SIV inactivated by aldrithiol-2 retained fusion activity similar to that seen with untreated virus. The relatively slow time course of SIV-cell fusion (t(1/2) = 19 min) indicates that the fusion events are stochastic. This feature provides a basis for understanding the mode of action of HIV/SIV entry inhibitors that target transition states.