Lipid rafts and HIV pathogenesis: host membrane cholesterol is required for infection by HIV type 1.

In a previous study we showed that budding of HIV-1 particles occurs at highly specialized membrane microdomains known as lipid rafts. These microdomains are characterized by a distinct lipid composition that includes high concentrations of cholesterol, sphingolipids, and glycolipids. Since cholesterol is known to play a key role in the entry of some other viruses, our observation of HIV budding from lipid rafts led us to investigate the role in HIV-1 entry of cholesterol and lipid rafts in the plasma membrane of susceptible cells. We have used 2-OH-propyl-beta-cyclodextrin (beta-cyclodextrin) to deplete cellular cholesterol and disperse lipid rafts. Our results show that removal of cellular cholesterol rendered primary cells and cell lines highly resistant to HIV-1-mediated syncytium formation and to infection by both CXCR4- and CCR5-specific viruses. beta-Cyclodextrin treatment of cells partially reduced HIV-1 binding, while rendering chemokine receptors highly sensitive to antibody-mediated internalization. There was no effect on CD4 expression. All of the above-described effects were readily reversed by incubating cholesterol-depleted cells with low concentrations of cholesterol-loaded beta-cyclodextrin to restore cholesterol levels. Cholesterol depletion made cells resistant to SDF-1-induced binding to ICAM-1 through LFA-1. Since LFA-1 contributes significantly to cell binding by HIV-1, this latter effect may have contributed to the observed reduction in HIV-1 binding to cells after treatment with beta-cyclodextrin. Our results indicate that cholesterol may be critical to the HIV-1 coreceptor function of chemokine receptors and is required for infection of cells by HIV-1.

Characterization of new syncytium-inhibiting monoclonal antibodies implicates lipid rafts in human T-cell leukemia virus type 1 syncytium formation.

We have previously shown that erythroleukemia cells (K562) transfected with vascular adhesion molecule 1 (VCAM-1) are susceptible to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation. Since expression of VCAM-1 alone is not sufficient to render cells susceptible to HTLV-1 fusion, K562 cells appear to express a second molecule critical for HTLV-induced syncytium formation. By immunizing mice with K562 cells, we have isolated four monoclonal antibodies (MAbs), K5.M1, K5.M2, K5.M3, and K5.M4, that inhibit HTLV-induced syncytium formation between infected MT2 cells and susceptible K562/VCAM1 cells. These MAbs recognize distinct proteins on the surface of cells as determined by cell phenotyping, immunoprecipitation, and Western blot analysis. Since three of the proteins recognized by the MAbs appear to be GPI linked, we isolated lipid rafts and determined by immunoblot analysis that all four MAbs recognize proteins that sort entirely or in large part to lipid rafts. Dispersion of lipid rafts on the cells by cholesterol depletion with beta-cyclodextrin resulted in inhibition of syncytium formation, and this effect was not seen when the beta-cyclodextrin was preloaded with cholesterol before treating the cells. The results of these studies suggest that lipid rafts may play an important role in HTLV-1 syncytium formation.

LuSIV cells: a reporter cell line for the detection and quantitation of a single cycle of HIV and SIV replication.

A single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIV(mac)239 LTR (-225 --> +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection.

CD4-independent, CCR5-dependent simian immunodeficiency virus infection and chemotaxis of human cells.

Most simian immunodeficiency virus (SIV), human immunodeficiency virus type 2 (HIV-2), and HIV-1 infection of host peripheral blood mononuclear cells (PBMCs) is CD4 dependent. In some cases, X4 HIV-1 chemotaxis is CD4 independent, and cross-species transmission might be facilitated by CD4-independent entry, which has been demonstrated for some SIV strains in CD4(-) non-T cells. As expected for CCR5-dependent virus, SIV required CD4 on rhesus and pigtail macaque PBMCs for infection and chemotaxis. However, SIV induced the chemotaxis of human PBMCs in a CD4-independent manner. Furthermore, in contrast to the results of studies using transfected human cell lines, SIV did not require CD4 binding to productively infect primary human PBMCs. CD4-independent lymphocyte and macrophage infection may facilitate cross-species transmission, while reacquisition of CD4 dependence may confer a selective advantage for the virus within new host species.

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.

Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts.

A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.

Increased infectivity of HIV type 1 particles bound to cell surface and solid-phase ICAM-1 and VCAM-1 through acquired adhesion molecules LFA-1 and VLA-4.

HIV-1 incorporates a variety of host membrane proteins during budding. We have previously shown that adhesion molecules are acquired by the virus in their activated or functional states. Our studies and those of others indicate that adhesion molecules can have profound effects on virus infectivity and its resistance to neutralization by antiviral antibodies. In this study we have examined the effect on infectivity of immobilization or margination of HIV-1 through acquired integrins LFA-1 and VLA-4 onto nonsusceptible cells and solid-phase adhesion ligands (ICAM-1 and VCAM-1, respectively). LFA-1- and VLA-4-mediated HIV-1 binding was supported by ICAM-1 and VCAM-1 immunoglobulin Fc chimeras, respectively. Integrin-mediated HIV-1 binding was also supported by 293 cells transfected with ICAM-1. In both cases the specificity of binding was confirmed with the appropriate blocking monoclonal antibodies or soluble adhesion ligands. We used a sensitive single-cycle infection assay based on a cell line expressing an LTR-luciferase cDNA construct to compare the infectivity of bound virus with that of free virus. Our results show that the binding of HIV-1 to nonsusceptible cells or immobilized adhesion ligands through acquired integrins can increase its infectivity by as much as two orders of magnitude. These results have implications for in vivo dissemination and transmission of HIV-1 and may also explain the high level of virus replication seen in solid lymphoid organs.

Production and characterization of monoclonal antibodies against pigtailed macaque (Macaca nemestrina) cell adhesion molecules.

Our previous in vitro studies indicate a significant role for cell adhesion molecules in the biology of HIV-1 and HTLV-1. Confirmation of the involvement of these molecules in the pathogenesis of retrovirus infection in vivo will require a suitable animal model. The SIV/pigtailed macaque (Macaca nemestrina) model of acquired immunodeficiency syndrome (AIDS) is an ideal system in which to study adhesion molecules and viral pathogenesis. The monoclonal antibodies (MAbs) against human adhesion molecules previously produced in our laboratory either do not react with or fail to block function of pigtailed macaque adhesion molecules. We have used papiovirus-transformed pigtailed macaque B cells as immunogen to generate murine MAbs against macaque adhesion molecules including ICAM-1, VCAM-1, and LFA-1. The specificity of the MAbs was confirmed by immunoprecipitation from lysates of vectorially iodinated cells, flow cytometry analysis of transfected cell lines and primary cells, binding assays on recombinant soluble human VCAM-1 and ICAM-1, and by inhibition of adhesion functions. MAbs against ICAM-1 and VCAM-1 showed positive staining of fixed tissue in immunohistochemistry studies. The same antibodies also blocked the function of these two adhesion molecules. The new MAbs can be used to study the tissue expression of adhesion molecules in SIV-infected animals as well as to test the involvement of these molecules in virus infection. Thus they should prove invaluable as probes of the role of cell adhesion molecules in AIDS pathogenesis in an animal model.

CD56 identifies monocytes and not natural killer cells in rhesus macaques.

BACKGROUND: CD56 is a lineage-specific marker of human natural killer (NK) cells. There are conflicts in the literature regarding the role of CD56 as a marker of NK cells in non-human primates. In the present study, we examined the role of CD56 in identifying rhesus NK cells. METHODS: The immunophenotype of normal macaque and human NK cells was analyzed by two- and three-color flow cytometry. Flow cytometric cell sorting was subsequently used to deplete or purify NK cells; the resulting cell populations were then used in standard chromium release assays of NK lytic function. RESULTS: In peripheral blood mononuclear cells of the rhesus macaque, CD56 was expressed primarily on cells with the light scatter and immunophenotypic profile of monocytes. Flow cytometric depletion of rhesus CD56(+) monocytic cells did not diminish functional activity against K562 cells, whereas depletion of CD8(+) or CD16(+) lymphocytes completely abrogated functional activity. Three-color flow cytometric analysis of CD8(+), CD16(+) lymphocytes showed that they expressed other markers (CD2, CD7, TIA-1) associated with NK cells, but notably, not CD56. CONCLUSIONS: These studies demonstrate that CD56 is not suitable as a marker of NK cells in the rhesus macaque.

The channel-forming toxin aerolysin neutralizes human immunodeficiency virus type 1.

Aerolysin is a channel-forming toxin secreted by Aeromonas spp. that binds to glycosyl phosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on sensitive target cells. Receptor binding is followed first by oligomerization of the toxin and then by insertion of the oligomers into the membrane to form stable channels that disrupt the permeability barrier. Human immunodeficiency virus type 1 (HIV-1) produced from T cells is known to incorporate Thy-1 and other GPI-anchored proteins into its membrane. Here, we show that aerolysin is capable of neutralizing HIV-1 in a dose-dependent manner and that neutralization depends upon the presence of these proteins in the viral envelope. Pretreatment with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins greatly reduced HIV-1 sensitivity to the toxin, and virus originating from a mutant cell line that lacks GPI-anchored proteins was not neutralized. Aerolysin variants with single amino acid changes that prevent oligomerization or insertion of the toxin were unable to inactivate the virus, implying that channel formation is necessary for neutralization to occur. These findings represent the first evidence that a pathogenic human virus can be neutralized by a bacterial toxin.

T cell-tropic HIV gp120 mediates CD4 and CD8 cell chemotaxis through CXCR4 independent of CD4: implications for HIV pathogenesis.

HIV entry is determined by one or more chemokine receptors. T cell-tropic viruses bind CXCR4, whereas macrophage-tropic viruses use CCR5 and other CCRs. Infection with CXCR4 and CCR5-tropic HIV requires initial binding to CD4, and chemotaxis induced by the CCR5-tropic envelope has been reported to be strictly dependent on CD4 binding. We demonstrate that, in contrast to CD4-dependent gp120 signaling via CCR5, envelope signaling through CXCR4 is CD4 independent, inducing chemotaxis of both CD4 and CD8 T cells. Signaling by virus or soluble envelope through CXCR4 may affect pathogenesis by attracting and activating target and effector cells.

Enhanced HIV type 1 neutralization by human anti-glycoprotein 120 monoclonal antibodies in the presence of monoclonal antibodies to lymphocyte function-associated molecule 1.

Cellular adhesion receptor LFA-1 and its ICAM ligands are known to play a role in HIV infection. The presence of these molecules on virions and target cells promotes virus infectivity and has previously been shown to hinder virus neutralization by anti-HIV antibodies. To delineate the effect of these molecules on neutralization of HIV-1, human monoclonal antibodies (MAbs) to V3 and the CD4-binding domain (CD4bd) of gp120 were examined in the presence of anti-LFA-1 MAbs. When either of two anti-LFA-1 MAbs was present, higher levels of virus neutralization were achieved by both anti-V3 and anti-CD4bd MAbs. This effect was observed with primary HIV-1 isolates as well as with a laboratory-adapted strain. However, this activity was seen only when an anti-LFA-1 MAb was combined with anti-gp120 MAbs that exhibited virus-specific neutralizing activities, demonstrating the specificity of both the anti-LFA-1 and anti-gp120 MAbs. Enhanced neutralization by anti-gp120 MAbs was observed if the anti-LFA-1 MAb was present during the initial 24 hr only, if added 24 hr after infection, or if present throughout the culture period. These data suggest that the anti-LFA-1 MAbs could act at different stages of HIV-1 infection, including the initial virus-cell interaction as well as during the amplification and spread of virus from cell to cell. These findings demonstrate the significant role of LFA-1 in HIV-1 infection and have important implications for evaluating the neutralizing activity of anti-HIV antibodies.

Antibody-dependent cell-mediated cytotoxicity can protect PBMC from infection by cell-associated HIV-1.

We have developed a novel in vitro assay system to study the role of antibody-dependent cell-mediated cytotoxicity (ADCC) in protection against HIV-1 infection by cell-associated virus. HIV-1-infected NK-resistant cells are mixed with specific antibody and unstimulated PBMC and ADCC is allowed to occur over several hours. The PBMC are then activated and cultured to allow virus replication in newly infected T cells. To ensure that ADCC is the only mechanism by which protection could occur we have used haptenated (TNP) infected cells and anti-hapten antibody. Anti-hapten sera completely protected PBMC from infection by haptenated HIV-1-infected cells in ADCC protection assays. F(ab')2 fragments of anti-hapten IgG showed no protection, confirming that ADCC was responsible for protection by anti-hapten IgG. PCR analysis for HIV-1 DNA confirmed the elimination of infected cells. We believe this to be the first direct demonstration that ADCC alone can protect PBMC from infection by cell-associated HIV-1.

Role of cellular adhesion molecules in HIV type 1 infection and their impact on virus neutralization.

While CD4 and several chemokine receptors are the principal receptors for human immunodeficiency virus type 1 (HIV-1) viruses, other cell membrane proteins also play a role in HIV-1 infection. A large array of host cell-derived membrane proteins, including adhesion molecules, are incorporated into the envelope of HIV-1 virions, and the profile of host cell proteins acquired by the virus depends on the cells used to propagate the virus. The major leukocyte adhesion molecules, such as leukocyte-function associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1), and CD44, retain their biological functions when expressed on the virion surface, and have been shown to increase virus-cell interaction, enhance virus infectivity, and extend the host cell range of the virus. LFA-1 and its ICAM ligands are also necessary for syncytium formation and cell-to-cell transmission of HIV-1. Furthermore, several studies demonstrate that the presence and level of cell-derived adhesion molecules on the surface of HIV-1 virions affect the process by which antibody-mediated virus neutralization occurs and is measured: the level of virus neutralization is influenced by the host cell-derived adhesion molecules present on the virus, and thus, by the type of host cells in which the virus was produced. Adhesion molecules expressed on the target cells used in neutralization assays similarly affect HIV-1 neutralization by virus-specific antibodies. Consistent with these observations is the finding that neutralizing activities of both HIV+ plasma and human anti-gp120 monoclonal antibodies (Mabs) are enhanced by an anti-LFA-1 Mab capable of blocking LFA-1 functions. Hence, LFA-1, ICAM-1, and other cellular adhesion molecules are involved in different stages of HIV-1 infection and profoundly affect HIV-1 neutralization by virus-specific antibodies. These findings illuminate the biology of virus-cell interactions and have significant implications for evaluating candidate HIV vaccines.

Syncytium-inhibiting monoclonal antibodies produced against human T-cell lymphotropic virus type 1-infected cells recognize class II major histocompatibility complex molecules and block by protein crowding.

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).

Immunoaffinity localization of the enzyme xanthine oxidase on the outside surface of the endothelial cell plasma membrane.

BACKGROUND: Reactive oxygen metabolites generated from endothelial xanthine oxidase (XO) trigger reperfusion injury in many organs. We evaluated the possibility that endothelial XO was localized on the endothelial cell surface, as well as within the cytoplasm. METHODS: Primary cultures of bovine (BAECs) and porcine (PAECs) aortic endothelial cells were grown in media documented to be free of XO. Polyclonal and monoclonal antibodies were developed against XO. These antibodies were used to evaluate BAEC and PAEC for cell surface XO through immunofluorescence staining, hybridoma cell surface labeling, and endothelial cell surface binding. RESULTS: These antibodies bound specifically to the surface of these cells when the membrane was shown to be intact and impermeable (and the cytoplasm inaccessible) to immunoglobulins Moreover, hybridoma cells expressing monoclonal antibody to XO bound specifically to the endothelial cell surface. Finally, intact endothelial cells bound specifically to the anti-XO polyclonal antibodies immobilized to the surface of a Petri dish. The integrity of these endothelial cell plasma membranes was demonstrated by the subsequent growth and replication of these cells in culture. CONCLUSIONS: These findings indicate that XO is present on the outside surface of the endothelial cell plasma membrane. This would not only explain the known in vivo efficacy of intravascularly administered large molecular weight antioxidants (such as superoxide dismutase) but could have important implications for inflammatory signaling.

Actin-dependent receptor colocalization required for human immunodeficiency virus entry into host cells.

Human immunodeficiency virus (HIV) envelope binds CD4 and a chemokine receptor in sequence, releasing hydrophobic viral gp41 residues into the target membrane. HIV entry required actin-dependent concentration of coreceptors, which could be disrupted by cytochalasin D (CytoD) without an effect on cell viability or mitosis. Pretreatment of peripheral blood mononuclear cells, but not virus, inhibited entry and infection. Immunofluorescent confocal microscopy of activated cells revealed CD4 and CXCR4 in nonoverlapping patterns. Addition of gp120 caused polarized cocapping of both molecules with subsequent pseudopod formation, while CytoD pretreatment blocked these membrane changes completely.

Human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation mediated by vascular cell adhesion molecule-1: evidence for involvement of cell adhesion molecules in HTLV-1 biology.

While studying the potential role of vascular cell adhesion molecule-1 (VCAM-1) in infection of endothelial cells by human immunodeficiency virus (HIV), we found that VCAM-1 can mediate human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. Both expression-vector-encoded and endogenously expressed VCAM-1 supported fusion of uninfected cells with HTLV-1-infected cells. Fusion was obtained with cell lines carrying the HTLV-1 genome and expressing viral proteins but not with an HTLV-1-transformed cell line that does not express viral proteins. In clones of VCAM-1-transfected cells, the degree of syncytium formation observed directly reflected the level of VCAM-1 expression. Syncytium formation between HTLV-1-expressing cells and VCAM-1+ cells could be blocked with antiserum against HTLV-1 gp46 and with a monoclonal antibody (MAb) against VCAM-1. Fusion was not blocked by antiserum against HIV or a MAb against VLA-4, the physiological counter-receptor for VCAM-1. The results indicate that VCAM-1 can serve as an accessory molecule or potential coreceptor for HTLV-1-induced cell fusion and provide direct evidence of a role for cell adhesion molecules in the biology of HTLV-1.

Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.

Antibody to human MHC class I inhibits SIVsmmPBj1.9-induced proliferation of pigtailed macaque lymphocytes.

Previously we have shown that the simian immunodeficiency virus SIVsmmPBj1.9, a molecular clone of SIVsmmPBj14, induces proliferation of human peripheral blood mononuclear cells (PBMC). We have extended this observation to show that SIVsmmPBj1.9 induces proliferation of PBMC from pigtailed macaques. This proliferative response was markedly inhibited by mAbs against human class I MHC, class II MHC and CD4 antigens, and partially inhibited by mAbs against integrin beta 2 subunit (CD18) and LFA-1 (CD11a). However, these antibodies differed in their ability to inhibit in vitro viral infectivity of PBMC. While anti-CD4, MHC class II, and LFA-1 strongly inhibited viral infectivity, antibodies to MHC class I demonstrated little effect on viral infectivity. A control antibody (PLM2) against porcine CD18 inhibited neither virus-induced proliferation nor viral infectivity. Based on these results, we suggest that SIVsmmPBj1.9-induced proliferation requires the participation of class I MHC, class II MHC and CD4 molecules. In addition, the observation that anti-class I MHC Ab inhibited proliferation of macaque PBMC induced by mitogen (PHA) and bacterial superantigens, such as Staphylococcus enterotoxin A and toxin shock syndrome toxin-1, suggests that SIVsmmPBj1.9 also contains a viral superantigen similar to that previously demonstrated in SIVsmmPBj14.