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In vitro experiments indicate that components of the host present in body fluids may prevent the attachment of human immunodeficiency virus type 1 (HIV-1) to target cells. Fibronectin (Fn), a dimeric 440-kDa extracellular matrix adhesion protein, is secreted by mesenchymal cells and assembled into insoluble matrices. Fn exerts important effects on cell growth and differentiation through a number of discrete functional domains. Several microorganisms are known to bind Fn. We show that, under physiological conditions, HIV-1 gp120 and gp160 are capable of binding plasma and cellular Fn as well as laminin and vitronectin. Experiments were set up to analyze in detail the binding of HIV gp120 and gp160 to Fn. The gp120 and gp160 specifically recognize the C-terminal heparin-binding domain of Fn (Fn-CTHBD) with a calculated KD of 2.8 x 10(-7) M for gp160. Binding of gp160 to Fn-CTHBD is a saturable and specific process that is blocked by antibodies to Fn-CTHBD and by heparin and is inhibited to a minor extent by heparan sulfate and dextran sulfate. These observations suggest that gp120/160 specifically recognize the III15 repeat within Fn-CTHBD. Intact Fn and Fn-CTHBD strongly inhibit the interaction of gp120/160 with soluble CD4 and, under low serum conditions, are capable of neutralizing the infectivity of HIV-1 for CD4-positive T cells. Thus, Fn that is present in plasma and mucinous secretions may well affect HIV infectivity and virus distribution in vivo.

The inhibitory effects on HIV replication of megalomicin (MGM, an inhibitor of intra-Golgi vesicle transport, have been studied. In experiments at low multiplicity of infection on Jurkat and MT2 cell lines. MGM inhibited the production of p24 antigen, the formation of syncytia, and the induction of apoptosis at concentrations below 5 microM. Furthermore, PCR analysis of genomic DNA showed that, in the presence of MGM, HIV-1 had been eradicated from the culture. MGM also inhibited replication of primary isolates of HIV-1 in blood lymphoblasts and more importantly, at 1 microM, MGM inhibited depletion of CD4+ T cells in cultures of blood lymphocytes from seropositive patients. Finally, MGM inhibited the generation of infectious virions and the processing of the envelope protein precursor gp160 to its mature forms, resulting in the rapid degradation of gp 160. These data suggest that MGM induces a powerful inhibitory effect on HIV-1 replication at nontoxic concentrations by preventing the processing of HIV-1 gp160 envelope protein and the subsequent formation of infectious viral particles.

We previously demonstrated that gp120/160 (Env) of HIV-1 interact in a carbohydrate-specific manner with mannosyl/N-acetylglucosaminyl derivatives and that HIV-1LAI infection of monocytic U937 and lymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactive glycopeptides from U937 cells. We report here that the natural glycoproteins bovine fetuin and asialofetuin, human orosomucoid and alpha-fetoprotein, and mannan, which all specifically interact with Env, inhibited infection of primary macrophages by macrophage-tropic HIV-1 strains, whereas dextran had no such effect. This activity was conserved if fetuin, asialofetuin, or orosomucoid were heat-treated, which rules out the role of their three-dimensional structure. Orosomucoid and mannan partially inhibited Env binding to macrophages but not to U937 or CEM cells. This indicates that Env does not bind in the same manner to primary macrophages and to immortalized CD4+ cells, and that orosomucoid and mannan act at CD4-independent stages of virus binding to macrophages. Mannan also inhibited Env binding to surface glycopeptides obtained after trypsin treatment of macrophages. Furthermore, orosomucoid and fetuin interacted with, and they inhibited the binding of a V3 loop B clade consensus peptide to several macrophage membrane proteins, including two 36 and 42 kDa proteins. These data indicate that these glycoproteins interfere with post-binding events during HIV-1 infection of primary macrophages. In contrast, the compounds did not affect infection of U937 or CEM cells by T-cell tropic HIV-1LAI nor infection of peripheral blood lymphocytes by HIV-1LAI or HIV-1(Ba-L). Thus, carbohydrate-specific inhibition of HIV infection depends on the cell type more than on the viral strain, and differences in the glycan structure of cell-type-specific cofactors may be important for HIV entry into cells.

Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site-specific photo-crosslinking we have followed the fate of the signal sequence of preprolactin in a cell-free system. This signal sequence has an unusually long hydrophilic n-region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N-terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV-1 envelope protein p-gp160 has the characteristic feature for calmodulin binding in its n-region. We found that a signal peptide fragment of p-gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now.

The human immunodeficiency virus type 1 (HIV-1) employs a number of complex strategies to interfere with the synthesis, stability, and subcellular localization of its specific cellular receptor CD4. To define better the mechanisms of inhibition of CD4 expression, we used a rabbit reticulocyte lysate in vitro system, in which cDNAs derived from HIV-1-infected cells were used to generate mRNA for the Tat, Vpu, and gp160 envelope proteins that were translated together with CD4-encoding mRNA. In the presence of microsomal membranes, we observed that cotranslation of Env mRNA resulted in a dose-dependent inhibition of CD4 translation. This effect was enhanced further when an mRNA-encoding Vpu in addition to Env mRNA was utilized. However, the activity of Vpu was mostly post-translational, since translation of Vpu alone, but not Env, was able to destabilize CD4 molecules presynthesized into microsomes. The Env-mediated inhibitory effect was specifically targeted at CD4 and did not affect the synthesis or stability of the CD8 molecule. Interestingly, mutated CD4 species, with a 20-fold lower affinity for HIV-1 Env than wild-type, were less sensitive to cotranslational inhibition. Our report identifies the envelope as the HIV-1 protein responsible for down-regulation of CD4 translation. We further propose a mechanism whereby direct interactions between gp160 and nascent CD4 molecules can cause interference with and premature termination of CD4 protein elongation.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) enter target cells by forming a complex between the viral envelope protein and two cell-surface membrane receptors: CD4 and a 7-span transmembrane chemokine receptor. Isolates of HIV that differ in cellular tropism use different subsets of chemokine receptors as entry cofactors: macrophage-tropic HIVs primarily use CCR5, whereas T-cell-tropic and dual-tropic isolates use CXCR4 receptors. HIV-mediated signal transduction through CCR5 is not required for efficient fusion and entry of HIV in vitro. Here we show that recombinant envelope proteins from macrophage-tropic HIV and SIV induce a signal through CCR5 on CD4+ T cells and that envelope-mediated signal transduction through CCR5 induces chemotaxis of T cells. This chemotactic response may contribute to the pathogenesis of HIV in vivo by chemo-attracting activated CD4+ cells to sites of viral replication. HIV-mediated signalling through CCR5 may also enhance viral replication in vivo by increasing the activation state of target cells. Alternatively, envelope-mediated CCR5 signal transduction may influence viral-associated cytopathicity or apoptosis.

CD4 molecules are the primary receptors for human immunodeficiency virus (HIV) and bind the envelope glycoprotein gp120 of HIV with high-affinity. We have previously shown that cross-linking of CD4 molecules (CD4XL) in normal peripheral blood mononuclear cells (PBMC) results in secretion of cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not of interleukin-2 (IL-2) or IL-4. To investigate the intracellular signaling events associated with CD4-gp120 interaction, we incubated CD4+ T cells from peripheral blood of HIV-negative healthy donors with HIV envelope protein gp160 alone or performed CD4XL with gp160 and anti-gp160 antibody. This procedure resulted in tyrosine phosphorylation of intracellular substrates p59fyn, zap 70, and p95vav and also led to ras activation, as assessed by conversion of rasGDP to rasGTP. The role of ras in CD4 signaling was further investigated using CD4+ Jurkat cells transfected with a dominant negative ras mutant. CD4+ T cells expressing dn-ras secreted significantly reduced levels of TNF-alpha in response to CD4XL. These studies indicate that interaction of HIV gp160 with CD4 molecules activates the ras pathway in T cells, which may result in the cells becoming unresponsive to subsequent stimulation.

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.

We generated replication-competent, recombinant vesicular stomatitis viruses (VSVs) expressing the human immunodeficiency virus (HIV) envelope protein or an HIV-VSV chimeric envelope protein in which the cytoplasmic domain of the HIV envelope protein was replaced with that from the VSV glycoprotein (G). These recombinants were generated with HIV type 1 (HIV-1) envelopes from both laboratory and primary isolates of HIV-1. The replication-competent recombinant viruses were stable and expressed the foreign proteins at high levels from extra transcription units in VSV. The foreign proteins were processed appropriately and transported to the cell surface. The incorporation of HIV gp120 into VSV particles was demonstrated biochemically only for the construct expressing the chimeric envelopes containing the VSV G cytoplasmic domain. The incorporation of the chimeric HIV envelope protein into the membrane of the recombinant VSV was also demonstrated by electron microscopy with gold-conjugated antibodies. To determine whether specific infection of CD4-positive cells could be demonstrated for these recombinants, we neutralized VSV infectivity due to VSV glycoprotein with anti-VSV serum. The neutralized recombinants expressing the chimeric envelope were able to infect only HeLa cells expressing CD4, and this CD4-specific infectivity was neutralized with anti-HIV serum. This assay also detected a 100-fold-lower titer of CD4-specific infectivity for the VSV recombinant expressing the wild-type HIV envelope. Our results illustrate that it is possible to express functional HIV envelopes from the VSV genome and target the recombinant virus to an alternative receptor. The recombinants may also prove useful as HIV vaccines.

In this study, specific signals known to mediate endoplasmic reticulum or Golgi localization of transmembrane proteins have been transferred to the human immunodeficiency virus type 1 (HIV-1) env gene product. The intracellularly retained recombinant glycoproteins were not proteolytically processed to gp120 and gp41, which is further evidence that this process occurs at a later stage in the transport pathway, presumably within or near the trans-Golgi network. Since the subcellular localization of the viral glycoproteins of enveloped viruses can be one of the factors determining the cellular site of particle assembly and release, experiments were performed to determine if this property was altered by coexpression of the recombinant HIV-1 glycoproteins. When wild-type virus was compared to mutant virus encoding the intracellularly retained glycoproteins, the extent of HIV-1 particle release into the extracellular medium remained unaffected, and electron-microscopic analysis did not reveal any significant alteration in the cellular sites of particle assembly and budding. Thus, in COS-7 cells, altered subcellular localization of the viral glycoprotein does not exert a dominant influence on the assembly site of the HIV-1 particle.

The intracellular proteolytic processing of HIV envelope glycoprotein gp160 into gp120/gp41 is an essential step for virus infectivity. Several convertases, belonging to the pro-protein convertase family, have been proposed as candidate gp160 processing enzymes. Here we demonstrate using RT-PCR that resting human T4 lymphocytes weakly express PC7, furin, and PC5 mRNA whereas lymphocytes activated under conditions favoring HIV replication express 5-10-fold higher levels of furin and PC7. In this report, we examined the capability of the newly cloned convertase PC7 to cleave gp160 into gp120/gp41 and compared it to furin. This was carried out in a cell-based assay whereby both gp160 and the cognate convertase were co-expressed in the constitutively secreting BSC40 cells and in the regulated AtT20 cells, as well as using two in vitro assays which examined the cleavage of gp160 or of a synthetic peptide spanning the cleavage site. The data demonstrate that PC7 can cleave specifically and in a cell-type specific manner gp160 into gp120gp41, suggesting that both furin and PC7 are so far the major PC-like candidate gp160 convertase in T4 lymphocytes.

We addressed the question of whether furin is the endoprotease primarily responsible for processing the human immunodeficiency virus type I (HIV-I) envelope protein gp160 in mammalian cells. The furin-deficient Chinese hamster ovary (CHO)-K1 strain RPE.40 processed gp160 as efficiently as wild-type CHO-K1 cells in vivo. Although furin can process gp160 in vitro, this processing is probably not physiologically relevent, because it occurs with very low efficiency. PACE4, a furin homologue, allowed processing of gp160 when both were expressed in RPE.40 cells. Further, PACE4 participated in the activation of a calcium-independent protease activity in RPE.40 cells, which efficiently processed the gp160 precursor in vitro. This calcium-independent protease activity was not found in another furin-deficient cell strain, 7.P15, selected from the monkey kidney cell line COS-7.

Ligands binding to the CD4 molecule can inhibit TCR-mediated T cell activation. We have previously reported that transcription factors regulating the expression of the IL-2 gene, NF-AT, NF-kappaB, and AP-1, are targets of this inhibitory effect in an in vitro model using peripheral human CD4+ T cells activated by a CD3 mAb. Two T cell activation pathways involved in the regulation of these transcription factors, calcium flux and the p21ras pathway, were investigated as potential targets. Binding of HIV envelope glycoprotein gp160/gp120 or a CD4 mAb to the CD4+ T cells, prior to TCR/CD3 activation, inhibited the intracellular calcium elevation. This event strongly suggested an inhibition of PLCgamma1 activity. Tyrosine phosphorylation of PLCgamma1, induced by CD3 activation, was not affected, but its association with tyrosine-phosphorylated proteins, including a 62-kDa protein, was disrupted. This PLCgamma1-associated p62 was found to be immunoreactive to p62-Sam68 Abs. The activation-induced phosphorylation of two p21ras effectors, Raf-1 and Erk2, was inhibited by the CD4 ligands, indirectly pointing to inhibition of the p21ras activation pathway. In addition, we demonstrate that TCR activation of normal CD4+ T cells induced the formation of p120GAP and PLCgamma1-containing complexes. These complexes also contain other unidentified proteins. CD4 ligand binding induced a defective formation of these transduction complexes. This may result in inefficient signaling, partially accounting for the inhibitory effects of the CD4 ligands on both p21ras and calcium-activation pathways.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays a major role in the down-regulation of its receptor, CD4. This down-regulation, at least in part, is caused by the formation of gp160-CD4 intracellular complexes which fail to transport out of the endoplasmic reticulum (ER). In this report, we have evaluated the ability of envelope glycoproteins from various isolates to block CD4 transport within the endoplasmic reticulum. Using a recombinant vaccinia virus expression system in HeLa cells, we expressed different HIV-1 and HIV-2 envelope glycoproteins with CD4. Pulse-chase labeling followed by immunoprecipitation demonstrated that envelope glycoproteins from primary and lab-adapted isolates were capable of forming intracellular complexes with CD4, resulting in the partial inhibition of CD4 transport to the Golgi. Although the efficiency of CD4 modulation was variable, these differences did not correlate with the type of isolate from which the HIV-1 glycoprotein was derived. However, we did find that the HIV-2 ST envelope glycoprotein (gp150) was not as efficient at blocking CD4 as the glycoprotein (gp140) derived from HIV-2 ROD. The decreased ability of ST gp150 to block CD4 within the ER was associated with an increased efficiency of ST gp150 transport and cleavage. Thus, differences in the ability of HIV envelope glycoproteins to block CD4 transport do exist, and these differences may be determined by envelope glycoprotein transport kinetics.

The human immunodeficiency virus HIV envelope glycoprotein gp160 is synthesized as an inactive precursor, which is processed into its fusiogenic form gp120/gp41 by host cell proteinases during its intracellular trafficking. Kexin/subtilisin-related endoproteases have been proposed to be enzyme candidates for this maturation process. In the present study, 1) we examined the ability of partially purified precursor convertases and their isoforms to cleave gp160 in vitro. The data demonstrate that all the convertases tested specifically cleave the HIV envelope glycoprotein into gp120 and gp41. 2) We demonstrated that a 19-amino acid model peptide spanning the gp120/gp41 junction is cleaved by all convertases at the same gp160 site as that recognized in HIV-infected cells. 3) In an effort to evaluate specific convertase inhibitors, we showed that the alpha1-antitrypsin variant, alpha1-PDX, inhibits equally well the ability of the tested convertases to cleave gp160 in vitro. 4) Three lymphocyte cell lines were screened by reverse transcription polymerase chain reaction in an effort to identify which are the convertases expressed in the most common HIV target, the CD4(+) lymphocytes. The data demonstrate that furin, PC5/6, and the newly cloned PC7 are the main transcribed convertases, suggesting that these proteinases are the major gp160-converting enzymes in T4 lymphocytes.

We have observed and analyzed an unexpected cross-reactivity of CD8+ CTL between two nonhomologous peptides of the HIV-1 IIIB gp160 envelope protein, P18 (residues 315-329) and HP53 (834-848, also called TH4.1), in the context of four different class I MHC molecules, Dd, Dp, Dq (or Lq), and H-2u. In strains expressing Dd, the cross-reactivity between peptides was bidirectional, whereas in other strains (H-2u, H-2p, and H-2q), the cross-reactivity was unidirectional; that is, P18-specific CTLs showed no killing against targets pulsed with HP53, although HP53 stimulated CTL showed cross-reactive lysis against P18-pulsed target cells. Cross-reactivity was also shown in immunization in vivo and with target cells endogenously expressing viral protein in vitro using two different recombinant vaccinia viruses expressing only the N-terminal portion of gp160, containing P18 but not HP53. Peptide cross-contamination was excluded. Cold target inhibition and single cell cloning experiments indicated that the same CTL was responding to both peptides. Using substituted and truncated peptides, we explored amino acid residues critical for cross-reactive CTL recognition, identified fine specificity similarities among all cross-reactive CTL lines but not non-cross-reactive lines, and mapped cross-reactivity to a 10-residue core of P18 and to an eight-residue core of HP53. A comparison of these peptide sequences and recent data on residues of P18 interacting with H-2Dd provided us with clues to residues involved in the interaction of the CTL with the MHC-peptide complex.

We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992). Infection of cloned H4/CD4 cells with the N1T strain of cell-free HIV-1 (HIV-1/N1T) was rapid and highly productive as measured by the initial expression of viral DNA, RNA, and protein, but all viral products declined to low levels by 14 days after infection. Chronically infected, virus-producing H4/CD4 cells could be obtained by cell cloning, indicating that HIV-1 DNA can integrate and remain expressed in these cells. The HIV-1 produced in H4/CD4 cells was noninfectious to glial cells, but it could be transmitted with low efficiency to CEM cells. Examination of viral protein composition by immunoprecipitation with AIDS serum or anti-gp120 antibody revealed that HIV-1/N1T-infected H4/CD4 cells produced all major viral proteins including gp160, but not gp120. Deglycosylation experiments with three different glycosidases determined that the absence of gp120 was not due to aberrant glycosylation of gp160, indicating a defect in gp160 proteolytic processing. Similar results were obtained in acutely and chronically infected H4/CD4 cells. To determine the generality of this HIV-1 replication phenotype in H4/CD4 cells, nine different viral clones were tested for replication in H4/CD4 cells by transfection. Eight were transiently productive like N1T, but one clone, NL4-3, established a long-lived productive infection in H4/CD4 cells, produced infectious progeny virus, and produced both gp160 and gp120. We conclude that for most HIV-1 strains tested, HIV-1 infection of H4/CD4 is restricted to a single cycle because of the defective processing of gp160, resulting in the absence of gp120 on progeny virus.

In order to better understand the role of the immunodominant V3 loop in the type-specific immune response and also to determine if this sequence has a role in AIDS pathogenesis, notably in the induction of apoptosis in CD4+ cells, we have introduced 2 modifications in the env gene from pNL4-3: a partial deletion in the V3 loop, keeping only the conserved tip of the loop GPGRAF consensus sequence (env delta V3-GPGRAF) and, secondly, a complete deletion of V3 sequence plus 43 nucleotides in C3 (env delta V3+). These constructions as well as the non-modified env gene, were cloned and expressed in a baculovirus system. Western blot analysis has shown that both modified env gene products reacted with a reference anti-HIV-1 serum to the same extent as the non-modified gp 160. However, in contrast to the non-modified env-protein and to env delta V3-GPGRAF, the env delta V3+ protein failed to bind to CD4 molecule, although V3 is not directly involved in receptor binding. These modified and non-modified recombinant proteins will be very useful to determine the potential of the partially or totally V3-deleted gp 160 to induce broadly reactive neutralizing antibodies and also to determine if V3 has a role in certain aspects of HIV-induced pathogenesis, notably apoptosis.

Classification of human immunodeficiency virus type 1 (HIV-1) by neutralization serotype may be important for the design of active and passive immunization strategies. Neutralizing antibody serotyping is hindered by the lack of standard reagents and assay format, and by the weak activity of many individual sera. To facilitate cross-clade neutralization analysis, we used an infectivity reduction assay (IRA) and selected clade-specific serum (or plasma) pools from subjects infected with clade B and E HIV-1, respectively. Several serum pools were utilized; some were selected for strong neutralizing activity against intraclade viruses and others were derived from conveniently available samples. Against a panel of 51 clade B and E viruses, serum pools displayed strong neutralization of most intraclade viruses and significantly diminished cross-clade neutralization. Results were confirmed against a blinded panel of 20 viruses. The data indicate that the phylogenetic classification of virus subtypes B and E corresponds to two distinct neutralization serotypes. This approach to neutralizing antibody serotyping may be useful in defining the antigenic relationship among viruses from other clades.

Specific interactions between the cell surface CD4 receptor and the HIV-1 envelope glycoprotein gp120 are responsible for the entry of HIV into host cells. Following infection, a down-modulation of CD4 at the cell surface is commonly observed. This may render cells resistant to subsequent infection by HIV as well as other viruses that also use CD4 as a portal of entry. This phenomenon is termed retroviral interference. CD4 down-modulation is complex and involves at least 3 viral gene products which include the envelope precursor gp160 and 2 auxilliary proteins Nef and Vpu. CD4 down-modulation has been observed in each of primary CD4+ T-lymphocytes and monocyte-derived macrophages, as well as both T and monocytic cell lines. CD4 down-regulation may occur at different levels. Specific binding of soluble gp120 may lead to internalization of CD4. The HIV-1 nef gene product which is expressed prior to HIV-1 structural proteins also causes the internalization of CD4 followed by its lysosomal degradation. During the late phase of viral gene expression i.e. viral structural protein synthesis, CD4-gp160 complexes forming in the ER represent another important factor leading to CD4 down-modulation. Finally, CD4 which is retained by gp160 in the ER, is specifically degraded in the presence of Vpu. Thus, it appears that CD4 down-regulation is of central importance to the life cycle of HIV-1.

To gain more insight into the processes leading to HIV-1 Env-induced cell death, we aim to coexpress stably wild-type and relevant mutant variants of both HIV-1 Env and human CD4 in lymphocyte cell lines. Here we report on the generation and characterization of several cell lines inducibly or constitutively expressing wild-type or cleavage-defective HIV-1 glycoproteins and human CD4 either singly or in combination. Coexpression of CD4 and wild-type Env led to the formation of multinucleated syncytia, to growth arrest and cell death, effects that all could be prevented by cultivation in the presence of monoclonal antibodies that inhibit cell surface membrane fusion. Cell lines coexpressing CD4 and mutated, noncleavable Env, detectable at the cell surface and still retaining CD4-binding capacity, were not retarded in their growth and cytolysis did not occur. These results indicate that cell lysis requires cell surface interaction of CD4 and gp120/41 and cleavage of gp160 to gp120 and gp41.

P18(IIIB) is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLA-A2.1 class I molecule is known to present P18(IIIB) for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV+ donors, a P18(IIIB)-specific CTL line was generated from and HLA-A2(+), HIV- donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, I10, with the sequence RGPGRAFVTI. Interestingly, this decamer is identical to the minimal epitope from P18(IIIB) seen by murine CTL restricted by H-2Dd. A panel of Ala-substituted peptides was employed in MHC-binding and T cell response studies to identify MHC- and TCR-binding residues. Notably, many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of I10 with the H-2Dd MHC molecule and murine I10-specific CTL. The I10 peptide does not contain the described HLA-A2.1 binding motif. Instead a Pro at P3, a Phe at P7 and an Ile at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein was expressed by a vaccinia vector that encodes the late promotor P11. The envelope protein synthesized mediated syncytia formation with SupT1 cells and was efficiently cleaved to produce mature gp120 and gp41 in CV-1 cells. gp 160 precursor processing was neither affected by a change in culture medium nor by a heterologous vesicular stomatitis virus coinfection. These results suggest that the proteolytic cleavage of gp160 is an efficient process and that coinfection with other viruses may not affect precursor processing of the HIV-1 Env protein.

We have previously demonstrated that human immunodeficiency virus (HIV) envelope glycoproteins have specific carbohydrate-binding properties for mannosyl/N-acetylglucosaminyl residues presented at high density on a carrier in vitro. Here, we investigated whether HIV envelope glycoprotein gp120 was able to interact with surface membrane carbohydrates of CD4+ cells by means of such lectin-carbohydrate interactions. CD4-free tryptic glycopeptides, prepared from the membrane of CD4+ monocytic U937 cells and partially purified by ConA-agarose affinity chromatography, could be eluted by mannan but not by methyl-alpha-mannose or methyl-alpha-glucose, which strongly suggests that they displayed oligomannosidic structures. These glycopeptides bound in a mannosyl-specific manner to radiolabeled recombinant gp120. Deglycosylation with N-glycanase which, as expected, strongly diminished binding of the glycopeptides to concanavalin A also abolished their interaction with gp120. In addition, the glycopeptides inhibited HIV infection of both U937 and CD4+ lymphoid CEM cells when preincubated with the virus. These findings indicate that, independently of the binding to CD4, mannosyl structures on CD4+ cells may play a role through lectin-carbohydrate interactions in envelope glycoprotein binding to a putative coreceptor(s) of HIV.