Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells.

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.     

High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells

Cell-to-cell virus transmission is one of the most efficient mechanisms of human immunodeficiency virus (HIV) spread, requires CD4 and coreceptor expression in target cells, and may also lead to syncytium formation and cell death. Here, we show that in addition to this classical coreceptor-mediated transmission, the contact between HIV-producing cells and primary CD4 T cells lacking the appropriate coreceptor induced the uptake of HIV particles by target cells in the absence of membrane fusion or productive HIV replication. HIV uptake by CD4 T cells required cellular contacts mediated by the binding of gp120 to CD4 and intact actin cytoskeleton. HIV antigens taken up by CD4 T cells were rapidly endocytosed to trypsin-resistant compartments inducing a partial disappearance of CD4 molecules from the cell surface. Once the cellular contact was stopped, captured HIV were released as infectious particles. Electron microscopy revealed that HIV particles attached to the surface of target cells and accumulated in large (0.5-1.0 microm) intracellular vesicles containing 1-14 virions, without any evidence for massive clathrin-mediated HIV endocytosis. The capture of HIV particles into trypsin-resistant compartments required the availability of the gp120 binding site of CD4 but was independent of the intracytoplasmic tail of CD4. In conclusion, we describe a novel mechanism of HIV transmission, activated by the contact of infected and uninfected primary CD4 T cells, by which HIV could exploit CD4 T cells lacking the appropriate coreceptor as an itinerant virus reservoir.     

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.     

Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55(Gag) in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55(Gag) in HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55(Gag) in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55(Gag) at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55(Gag) and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55(Gag) with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.     

HIV gp120-induced interaction between CD4 and CCR5 requires cholesterol-rich microenvironments revealed by live cell fluorescence resonance energy transfer imaging

Binding of the human immunodeficiency virus (HIV) envelope gp120 glycoprotein to CD4 and CCR5 receptors on the plasma membrane initiates the viral entry process. Although plasma membrane cholesterol plays an important role in HIV entry, its modulating effect on the viral entry process is unclear. Using fluorescence resonance energy transfer imaging, we have provided evidence here that CD4 and CCR5 localize in different microenvironments on the surface of resting cells. Binding of the third variable region V3-containing gp120 core to CD4 and CCR5 induced association between these receptors, which could be directly monitored by fluorescence resonance energy transfer on the plasma membrane of live cells. Depletion of cholesterol from the plasma membrane abolished the gp120 core-induced associations between CD4 and CCR5, and reloading cholesterol restored the associations in live cells. Our studies suggest that, during the first step of the HIV entry process, gp120 binding alters the microenvironments of unbound CD4 and CCR5, with plasma membrane cholesterol required for the formation of the HIV entry complex.     

V3 loop-derived peptide SPC3 inhibits infection of CD4- and galactosylceramide- cells by LAV-2/B.

SPC3, a synthetic multibranched peptide including the GPGRAF consensus motif of the human immunodeficiency virus type 1 (HIV-1) gp120 V3-loop is a potent inhibitor of HIV infection of human CD4+ lymphocytes, macrophages and CD4-/galactosylceramide+ human colon epithelial cells and is currently tested in phase II clinical trials (FDA protocol 257 A). The antiviral property of SPC3 was further investigated for its ability to inhibit LAV-2/B, an HIV-2 clone with a CD4-independent tropism. SPC3 inhibited the LAV-2/B-mediated infection of B-cell line which does not express the CD4 and the galactosylceramide molecules on their cell surface, suggesting an SPC3-sensitive CD4/galactosylceramide-independent pathway of viral infection in HIV susceptible cells. The molecular mechanism of the peptide inhibition was also investigated. The data suggested that the SPC3-mediated inhibition does not result from a direct competition between SPC3 and gp120 binding to the cell surface of the target cell.     

Characterization of the tertiary structure of soluble CD4 bound to glycosylated full-length HIVgp120 by chemical modification of arginine residues and mass spectrometric analysis

The initial step of infection of blood cells with the human immunodeficiency virus, HIV, is the formation of a complex of the viral envelope protein gp120 and its human receptor CD4. We have examined structural features of recombinant soluble CD4 (sCD4) by chemical modification of arginine residues with hydroxyphenylglyoxal and subsequent analysis by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. As R58, R59, R131, R134, R219, R240, R293, and R329 could be derivatized free in solution, these arginine residues were exposed on the surface of the protein. In the noncovalent complex of sCD4 with HIV(SF2)gp120, only R58, R131, R134, R219, R240, R293, and R329 were accessible for the derivatizing agent. R59 was shielded from hydroxyphenylglyoxal and was, therefore, considered to be part of the interaction site with gp120. This indicates that the carbohydrate moieties and the flexible variable loops of the glycosylated full-length gp120 from HIV strain SF2 do not induce a reorganization of CD4 in its binding to gp120 and, therefore, do not appear to significantly affect the structural orientation of the primary receptor in complex with the HIV envelope protein as compared to the binding observed in the crystal structure of CD4 with truncated deglycosylated gp120.     

Partial activation and induction of apoptosis in CD4(+) and CD8(+) T lymphocytes by conformationally authentic noninfectious human immunodeficiency virus type 1

Increased levels of apoptosis are seen in human immunodeficiency virus (HIV) infection, and this has been proposed as an important mechanism contributing to HIV pathogenesis. However, interpretation of in vitro studies aimed at understanding HIV-related apoptosis has been complicated by the use of high concentrations of recombinant proteins or by direct cytopathic effects of replicating virus. We have developed an inactivation procedure that destroys retroviral infectivity while preserving the structural and functional integrity of the HIV surface proteins. These noninfectious virions interact authentically with target cells, providing a powerful tool to dissect mechanisms of HIV pathogenesis that do or do not require viral replication. Noninfectious CXCR4-tropic HIV-1 virions, but not microvesicles, partially activated freshly isolated CD4(+) and CD8(+) peripheral blood mononuclear cell T lymphocytes to express FasL and Fas, but not CD69 or CD25 (interleukin-2 receptor alpha) and eventually die via apoptosis starting 4 to 6 days postexposure. These effects required conformationally intact virions, as heat-denatured virions or equivalent amounts of recombinant gp120 did not induce apoptosis. The maximal apoptotic effect was dependent on major histocompatibility complex (MHC) class II proteins being present on the virion, but was not MHC restricted. The results suggest that the immunopathogenesis of HIV infection may not depend solely on direct cytopathic effects of HIV replication, but that effects due to noninfectious HIV-1 virions may also contribute importantly.     

Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity.

We constructed a recombinant human immunodeficiency virus (HIV) vector to facilitate studies of virus infectivity. A drug resistance gene was inserted into a gp160- HIV proviral genome such that it could be packaged into HIV virions. The HIV genome was rendered replication defective by deletion of sequences encoding gp160 and insertion of a gpt gene with a simian virus 40 promoter at the deletion site. Cotransfection of the envelope-deficient genome with a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. The drug resistance gene was transmitted and expressed upon infection of susceptible cells, enabling their selection in mycophenolic acid. This system provides a quantitative measure of HIV infection, since each successful infection event leads to the growth of a drug-resistant colony. The HIV-gpt virus produced was tropic for CD4+ human cells and was blocked by soluble CD4. In the absence of gp160, noninfectious HIV particles were efficiently produced by cells transfected with the HIV-gpt genome. These particles packaged HIV genomic RNA and migrated to the same density as gp160-containing virions in a sucrose gradient. This demonstrates that HIV virion formation is not dependent on the presence of a viral envelope glycoprotein. Expression of a murine leukemia virus amphotropic envelope gene in cells transfected with HIV-gpt resulted in the production of virus capable of infecting both human and murine cells. These results indicate that HIV can incorporate envelope glycoproteins other than gp160 onto particles and that this can lead to altered host range. Like HIV type 1 and vesicular stomatitis virus(HIV) pseudotypes, gp-160+ HIV-gpt did not infect murine NIH 3T3 cells that bear human CD4, confirming that these cells are blocked at an early stage of HIV infection.     

Dextran sulfate blocks antibody binding to the principal neutralizing domain of human immunodeficiency virus type 1 without interfering with gp120-CD4 interactions.

The mechanism of the antiviral activity of sulfated polysaccharides on human immunodeficiency virus type 1 (HIV-1) was investigated by determining the effect of dextran sulfate on the binding of CD4 and several anti-gp120 monoclonal antibodies to both recombinant and cell surface gp120. Dextran sulfate did not interfere with the binding of sCD4 to rgp120 on enzyme-linked immunosorbent assay (ELISA) plates or in solution and did not block sCD4 binding to HIV-1-infected cells expressing gp120 on the cell surface. Dextran sulfate had minimal effects on rgp120 binding to CD4+ cells at concentrations which effectively prevent HIV replication. In contrast, it potently inhibited the binding of both rgp120 and cell surface gp120 to several monoclonal antibodies directed against the principal neutralizing domain of gp120 (V3). In an ELISA format, dextran sulfate enhanced the binding of monoclonal antibodies against amino-terminal regions of gp120 and had no effect on antibodies directed to other regions of gp120, including the carboxy terminus. The inhibitory effects of polyanionic polysaccharides on viral binding, viral replication, and formation of syncytia therefore appear mediated by interactions with positively charged amino acids concentrated in the V3 region. This high local positive charge density, unique to the V3 loop, leads us to propose that this property is critical to the function of the V3 region in mediating envelope binding and subsequent fusion between viral and cell membranes. The specific interaction of dextran sulfate with this domain suggests that structurally related molecules on the cell surface, such as heparan sulfate, may be additional targets for HIV binding and infection.     

A biochemical approach for detecting interactions between peptides from the HIV gp120 glycoprotein and a CD4 sequence

Peptides selected from the HIV viral protein gp120 bind to a synthetic peptide mimicking sequence 78-89 of the human lymphocyte CD4 molecule, linked to activated Sepharose. The binding of viral fragments to the CD4 peptide-Sepharose beads was ascertained either by aid of a ninhydrin reagent or by fluorescence microscopy. A suitable alignment of these HIV peptides with the CD4 fragment showed that multiple interactions might occur between hydrophobic or charged groups of the two molecules. Although this experiment does not demonstrate that these two amino acid stretches are involved in the primary binding of gp120 to CD4 receptors, the present data suggest that the two sequences might have some kind of interaction during subsequent steps of viral infection.     

Expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-CD4 receptor complex

The infection of CD4(+) host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.     

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.     

Surfactant protein A binds to HIV and inhibits direct infection of CD4+ cells, but enhances dendritic cell-mediated viral transfer

The identification of surfactant protein A (SP-A) as an important innate immune factor of the lungs, amniotic fluid, and the vaginal tract suggests that it could play an important role during various stages of HIV disease progression and transmission. Therefore, we examined whether SP-A could bind to HIV and also had any effect on viral infectivity. Our data demonstrate that SP-A binds to HIV in a calcium-dependent manner that is inhibitable by mannose and EDTA. Affinity capture of the HIV viral lysate reveals that SP-A targets the envelope glycoprotein of HIV (gp120), which was confirmed by ELISA using recombinant gp120. Digestion of gp120 with endoglycosidase H abrogates the binding of SP-A, indicating that the high mannose structures on gp120 are the target of the collectin. Infectivity studies reveal that SP-A inhibits the infection of CD4+ T cells by two strains of HIV (BaL, IIIB) by >80%. Competition assays with CD4 and mAbs F105 and b12 suggest that SP-A inhibits infectivity by occlusion of the CD4-binding site. Studies with dendritic cells (DCs) demonstrate that SP-A enhances the binding of gp120 to DCs, the uptake of viral particles, and the transfer of virus from DCs to CD4+ T cells by >5-fold at a pH representative of the vaginal tract. Collectively, these results suggest that SP-A acts as a dual modulator of HIV infection by protecting CD4+ T cells from direct infection but enhancing the transfer of infection to CD4+ T cells mediated by DCs.     

Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.     

Truncated variants of gp120 bind CD4 with high affinity and suggest a minimum CD4 binding region.

The envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) binds the cellular protein CD4 with high affinity. By deletion we show that 62 N- and 20 C-terminal residues along with the V1, V2 and V3 variable regions of gp 120 are unnecessary for CD4 binding. A 287 residue variant (ENV59), missing those 197 amino acids, binds to CD4 with high affinity. A polyclonal antibody failed to efficiently precipitate ENV59 which is consistent with the loss of immunodominant antigenic structures in the regions deleted. This suggests that ENV59 may have potential as an immunogen, able to elicit antibodies against more conserved regions of gp120. Additionally, complementing co-expressed gp120 fragments as well as a circularly permuted molecule bind CD4, and suggest either that the molecular termini are adjacent in the folded structure, or that an N-terminal region folds into the structure unconstrained by its method of attachment to the rest of the molecule.     

Phorbol ester-induced down modulation of tailless CD4 receptors requires prior binding of gp120 and suggests a role for accessory molecules.

The entry of human immunodeficiency virus type 1 into cells proceeds via a fusion mechanism that is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. Species- and tissue-specific restrictions to viral entry suggested the participation of additional membrane components in the postbinding fusion events. In a previous study (H. Golding, J. Manischewitz, L. Vujcic, R. Blumenthal, and D. Dimitrov, J. Virol. 68:1962-1968, 1994), it was found that phorbol myristate acetate (PMA) inhibits human immunodeficiency virus type 1 envelope-mediated cell fusion by inducing down modulation of an accessory component(s) in the CD4-expressing cells. The fusion inhibition was seen in a variety of cells, including T-cell transfectants expressing engineered CD4 receptors (CD4.401 and CD4.CD8) which are not susceptible to down modulation by PMA treatment. In the current study, it was found that preincubation of A2.01.CD4.401 cells with soluble monomeric gp120 for 1 h at 37 degrees C primed them for PMA-induced down modulation (up to 70%) of the tailless CD4 receptors. The gp120-priming effect was temperature dependent, and the down modulation may have occurred via clathrin-coated pits. Importantly, nonhuman cell lines expressing tailless CD4 molecules did not down modulate their CD4 receptors under the same conditions. The gp120-dependent PMA-induced down modulation of tailless CD4 receptors could be efficiently blocked by the human monoclonal antibodies 48D and 17B, which bind with increased avidity to gp120 that was previously bound to CD4 (M. Thali, J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski, J. Virol. 67:3978-3988, 1993). These findings suggest that gp120 binding to cellular CD4 receptors induces conformational changes leading to association of the gp120-CD4 complexes with accessory transmembrane molecules that are susceptible to PMA-induced down modulation and can target the virions to clathrin-coated pits.     

Atomic force microscopy investigation of human immunodeficiency virus (HIV) and HIV-infected lymphocytes

Isolated human immunodeficiency virus (HIV) and HIV-infected human lymphocytes in culture have been imaged for the first time by atomic force microscopy (AFM). Purified virus particles spread on glass substrates are roughly spherical, reasonably uniform, though pleomorphic in appearance, and have diameters of about 120 nm. Similar particles are also seen on infected cell surfaces, but morphologies and sizes are considerably more varied, possibly a reflection of the budding process. The surfaces of HIV particles exhibit "tufts" of protein, presumably gp120, which do not physically resemble spikes. The protein tufts, which number about 100 per particle, have average diameters of about 200 A, but with a large variance. They likely consist of arbitrary associations of small numbers of gp120 monomers on the surface. In examining several hundred virus particles, we found no evidence that the gp120 monomers form threefold symmetric trimers. Although >95% of HIV-infected H9 lymphocytic cells were producing HIV antigens by immunofluorescent assay, most lymphocytes displayed few or no virus on their surfaces, while others were almost covered by a hundred or more viruses, suggesting a dependence on cell cycle or physiology. HIV-infected cells treated with a viral protease inhibitor and their progeny viruses were also imaged by AFM and were indistinguishable from untreated virions. Isolated HIV virions were disrupted by exposure to mild neutral detergents (Tween 20 and CHAPS) at concentrations from 0.25 to 2.0%. Among the products observed were intact virions, the remnants of completely degraded virions, and partially disrupted particles that lacked sectors of surface proteins as well as virions that were split or broken open to reveal their empty interiors. Capsids containing nucleic acid were not seen, suggesting that the capsids were even more fragile than the envelope and were totally degraded and lost. From these images, a good estimate of the thickness of the envelope protein-membrane-matrix protein outer shell of the virion was obtained. Treatment with even low concentrations (<0.1%) of sodium dodecyl sulfate completely destroyed all virions but produced many interesting products, including aggregates of viral proteins with strands of nucleic acid.