Inhibit Human Immunodeficiency Virus Infection Research Articles

The Anti-HIV Pentameric Pseudopeptide HB-19 Binds the C-terminal End of Nucleolin and Prevents Anchorage of Virus Particles in the Plasma Membrane of Target Cells

The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.

PMTI, a broadly active unusual single-stranded polyribonucleotide, inhibits human immunodeficiency virus replication by multiple mechanisms.

Poly(1-methyl-6-thioinosinic acid), or PMTI, is a single-stranded polyribonucleotide and is the first homopolyribonucleotide devoid of Watson-Crick hydrogen bonding sites to show potent human immunodeficiency virus (HIV) inhibition. PMTI was found to be active when evaluated against a variety of low passage clinical HIV isolates in fresh human peripheral blood cells, including T cell-tropic and monocyte-macrophage-tropic viruses, syncytium-inducing and non-syncytium-inducing viruses and viruses representative of the various HIV-1 clades (A through F). The compound was active against HIV-2, all nucleoside and non-nucleoside reverse transcriptase (RT) inhibitor drug-resistant virus isolates tested and interacted with AZT or ddl to synergistically inhibit HIV infection. In biochemical inhibition assays, PMTI was determined to be a potent inhibitor of HIV-1 and HIV-2 RT, including RTs with mutations that engender resistance to nucleoside and non-nucleoside RT inhibitors. PMTI inhibited both the polymerase and RNase H activities of HIV RT. PMTI did not inhibit HIV-1 protease or integrase. Cell-based mechanism of action assays indicated that PMTI also interfered with early events in the entry of HIV into target cells. Furthermore, PMTI inhibited the fusion of gp120-expressing and CD4-expressing cells, but at concentrations approximately 1 log10 greater than those that inhibited virus entry. These results suggest that the homopolyribonucleotide PMTI blocks HIV replication in human cells at its earliest stages by multiple mechanisms, inhibition of virus entry and inhibition of RT.

An Anti-HIV Peptide Construct Derived from the Cleavage Region of the Env Precursor Acts on Env Fusogenicity through the Presence of a Functional Cleavage Sequence

A 22-amino-acid-long multibranched peptide construct (CLV) derived from the cleavage region (KIEPLGVAPTKAKRR*VVQREKR*) of the human immunodeficiency virus (HIV) type-1 envelope precursor inhibits HIV infection (Virology,1996, 223, 406–408). We attempted to characterize its activity for Env expressedviaa recombinant vaccinia virus (rVV): gp160 cleavage was delayed, but not impaired, in the presence of CLV (10 μM), whereas neither Env production nor Env membrane expression was significantly altered. Through the synthesis of analogs, we concluded that the presence of a cleavage sequence was required for inhibition of syncytium formation by CLV in rVV-infected CD4+cell cultures: indeed, a single amino acid residue substitution (R*>S) in the cleavage sites presented by CLV abolished its activity. Other analogs allowed us to further determine the region of CLV which mediates its activity. The ability of a radiolabeled CLV analog to enter cells was also shown. Altogether, these data strongly suggest that CLV acts on Env fusogenicity at least partially through interference with gp160 processing.

Productive Human Immunodeficiency Virus-1 Infection of Purified Megakaryocytic Progenitors/Precursors and Maturing Megakaryocytes

AbstractWe have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the following approach: (1) human hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood and grown in serum-free liquid suspension culture supplemented with thrombopoietin (Tpo), generated a relatively large number of ≥ 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1–challenged cells do not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV transcripts and proteins were clearly detected in all NL4-3 infection experiments by RT-PCR and p24 assay, respectively, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV+ MKs varies from at least 1% and 5% for day 0 and day 5 infected cells, respectively. Production of an infectious viral progeny, evaluated by the capability of culture supernatants from day 5 NL4-3–challenged MKs to infect C8166 T-lymphoblastoid cell line, was consistently observed (viral titer, ≈ 5 × 103 tissue culture infectious dose50/mL/106 cells). Exposure of MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody (MoAb), which recognizes the CD4 region binding with the gp120 envelope glycoprotein, markedly inhibited HIV infection, as indicated by a reduction of p24 content in the supernatants: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alternative mechanism of viral entry may exist. Morphologic analysis of day 12 MKs derived from HPCs infected at day 0 showed an impaired megakaryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that ≈ 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megakaryocytic precursors challenged at day 5 to 8 generated fully mature day 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different days of culture, showed an impaired megakaryocytopoiesis comparable to that observed in HIV-infected cells, thus suggesting that Tat is a major mediator in the above described phenomena. These results shed light on the pathogenesis of HIV-related thrombocytopenia; furthermore, they provide a model to investigate the effects of HIV on megakaryocytic differentiation and function.

Pseudopeptide TASP Inhibitors of HIV Entry Bind Specifically to a 95-kDa Cell Surface Protein

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KPsi(CH2N)PR]-TASP construct, Psi(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KPsi(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KPsi(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KPsi(CH2N)PR]-TASP, the p95.TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KPsi(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KPsi(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.

Role of CD4 epitopes outside the gp120-binding site during entry of human immunodeficiency virus type 1.

CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus.

Membrane glycolipids and human immunodeficiency virus infection of primary macrophages.

The membrane glycolipids galactosylceramide (GalCer) and sulfatide (SGalCer) have been reported to act as receptors of human immunodeficiency virus (HIV) on CD4- cell lines. We show here that these glycolipids are present on CD4+ cells purified from human blood and on in vitro-differentiated monocyte-derived macrophages (MDMs). We investigated the role they could play in HIV infection. Glycolipids of MDMs were characterized at the molecular level by immunolabeling and thin-layer chromatography immune overlay, using a panel of human-, rabbit-, or murine-specific antibodies. GalCer and SGalCer were expressed at the surface of MDMs as assessed by indirect immunofluorescence and flow cytometry analysis, and they could be characterized with specific antibodies in the cellular glycolipid extracts in addition to GM1, GM3, and GD1b gangliosides. Recombinant 125-I-labeled gp160 specifically bound to GalCer, SGalCer, GM1, and GM3 as well as to phospholipids (phosphatidylethanolamine and phosphatidylserine) from MDM extracts. Anti-SGalCer monoclonal antibodies (MAbs), but not anti-GalCer antibodies, entailed limited (30-40%) but significant inhibition of gp160 binding to MDMs. However, the four human anti-SGalCer MAbs and the three murine or rabbit ant-GalCer antibodies tested did not inhibit HIV infection of MDMs, in contrast to CD4 antibody anti-Leu3a tested in parallel. These findings suggests that although HIV envelope glycoprotein can bind to SGalCer and GalCer from CD4+ MDM extracts, these glycolipids do not apparently act as HIV coreceptors nor are they involved in HIV infection of these cells.

Similar Structural Models of the Transmembrane Proteins of Ebola and Avian Sarcoma Viruses

Similar Structural Models of the Transmembrane Proteins of Ebola and Avian Sarcoma Viruses

Inhibition of human immunodeficiency virus infection of CD4+ cells by CD4-free glycopeptides from monocytic U937 cells.

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.

Inhibition of in vitro HIV infection by trinitrophenyl-protein conjugates

Levels of natural antibodies (NAb) with high anti-trinitrophenyl (TNP) activity are increased during human immunodeficiency virus (HIV) infection. The aim of the present study was to examine the anti-HIV effect of natural anti-TNP antibodies, as well as that of their internal image, TNP antigen, on HIV infection in vitro. The results obtained with anti-TNP antibodies, as assessed by syncytia formation, were variable, although they demonstrated an inhibitory effect. In contrast, using RT activity assay plus evaluation of syncytia formation and the viral cytopathic effect, we found that bovine serum albumin (BSA) bearing different TNP groups was able to inhibit HIV infection of peripheral mononuclear cells and T4 cell lines without affecting cell metabolism or proliferation. BSA alone was devoid of activity; the antiviral effect depended on TNP substitution of the BSA molecule, and passage through an anti-TNP immunoadsorbent abolished this effect. The mechanism by which TNP exerts this antiviral effect is unclear. Antigenic epitopes may be shared by HIV and TNP, since monoclonal antibodies directed against various HIV proteins reacted with TNP in an enzyme immunoassay. TNP-BSA, however, did not bind to the CD4 receptor.

Gene therapy in the treatment of disease

Gene therapy or transfer is increasingly used as a therapeutic modality in diseases that span the medical spectrum. The American Academy of Allergy and Immunology and the National Institute of Allergy and Infectious Diseases jointly sponsored the Symposium, "Gene Therapy in the Treatment of Disease," which was held December 6, 1993 at the National Institutes of Health (NIH). The program committee consisted of Lawrence Lichtenstein, MD, PhD, (Baltimore) Chairman; Philip Fireman, MD, (Pittsburgh); Howard B. Dickler, MD, (Bethesda); William Paul, MD, (Bethesda); and Raif Geha, MD, (Boston). Eight outstanding presentations on current and potential uses of gene therapy were provided by speakers who are leading researchers in this area of broad interest and unusually promising clinical application. This report summarizes the highlights of those presentations.

A peptide inhibitor of human immunodeficiency virus infection binds to novel human cell surface polypeptides

Putative cell surface human immunodeficiency virus (HIV) gp41 receptor proteins of 45 and 80 kDa (p45 and p80, respectively) were identified on human cells using a 17-amino acid peptide, referred to as CS3. In contrast, murine P815 cells expressed a peptide binding protein of 80 kDa only. A segment of 8 amino acids within CS3 contains the minimum sequence able to inhibit binding of radiolabeled CS3 to p80 and p45, as shown by competitive binding studies. Human p45 was purified from CD4+ RH9 cells by CS3 peptide affinity chromatography. Human p80 was partially purified from RH9 cell lysates by size exclusion chromatography followed by SDS-polyacrylamide gel electrophoresis; a rabbit polyclonal antibody was raised against this preparation. Anti-p80 antibody inhibited HIV infection in a dose-dependent manner. The CS3 region of gp41 has been been shown previously to be exposed on viral particles and envelope-expressing cells predominately after conformational changes in the HIV envelope occur due to the interaction of CD4 with gp120. These results, together with those from previous studies, suggest that following the interaction of gp120 with CD4, there may be a second receptor interaction necessary for virus entry/fusion.

RETROVIRUS INFECTION OF ENTEROCYTES

SummaryAlthough the gastrointestinal tract is thought to be one of the major routes for infection with human immunodeficiency virus (HIV), controversy still exists concerning the ability of HIV to infect epithelial cells of the intestinal mucosa. In this paper, Fantini et al. detailed their studies of the ability of HIV to infect the human colon cancer cell line HT‐29‐D4. Differentiation of these epithelial cells can be regulated in vitro by modifying the culture medium concentration of glucose and by replacing glucose with galactose. Intestinal cells in culture were infected with three different strains of HIV—HIV‐1‐BRU, HIV‐1‐NDK (a highly cytopathic strain), and HIV‐2‐ROD. Exponentially growing HT‐29‐D4 cells were exposed for 16 h to HIV in serum‐supplemented medium. After washing, epithelial cells were harvested and subcultured. Virus replication in subcultured cells was determined by HIV‐specific DNA amplification using the polymerase chain reaction. Amplified products were analyzed for specificity by Southern blot hybridization with a radiolabeled DNA probe internal to the primer templates. The presence of CD4 molecules (i.e., receptors for cell binding of HIV) on intestinal epithelial cells was assessed using monoclonal antibodies and immunofluorescence labeling.Fantini et al. found that undifferentiated HT‐29‐D4 cells express a CD4‐related antigen that lacks the epitope known to be involved in recognition of the HIV envelope binding protein referred to as gp120. Undifferentiated HT‐29‐D4 cells were infected by both HIV‐1 and HIV‐2 strains. However, the infection did not alter cell growth. Differentiated HT‐29‐D4 cells were also infected, either through the apical brush border or the basolateral membrane. Although the CD4‐like surface molecules were restricted to the basolateral domain of differentiated cells, antibodies against specific epitopes of the CD4 glycoprotein did not inhibit HIV infection. The authors conclude that intestinal epithelial cells can be infected by both HIV‐1 and HIV‐2 via either the apical membrane or the basolateral membrane. There was no evidence of viral replication by reverse transcriptase activity. However, following culture of HT‐29‐D4 cells with virus, HIV DNA could be recovered by gene amplification as long as 9 months after exposure of intestinal tissue culture cells to the virus. The authors speculate that this might provide an explanation for the prolonged seronegative state observed in HIV‐infected individuals.These findings raise the possibility that infected lamina propria lymphocytes and macrophages could coinfect intestinal epithelial cells. In addition, HIV present in the intestinal lumen could bind to the apical surface and infect enterocytes.

RETROVIRUS INFECTION OF ENTEROCYTES

Summary Although the gastrointestinal tract is thought to be one of the major routes for infection with human immunodeficiency virus (HIV), controversy still exists concerning the ability of HIV to infect epithelial cells of the intestinal mucosa. In this paper, Fantini et al. detailed their studies of the ability of HIV to infect the human colon cancer cell line HT-29-D4. Differentiation of these epithelial cells can be regulated in vitro by modifying the culture medium concentration of glucose and by replacing glucose with galactose. Intestinal cells in culture were infected with three different strains of HIV—HIV-1-BRU, HIV-1-NDK (a highly cytopathic strain), and HIV-2-ROD. Exponentially growing HT-29-D4 cells were exposed for 16 h to HIV in serum-supplemented medium. After washing, epithelial cells were harvested and subcultured. Virus replication in subcultured cells was determined by HIV-specific DNA amplification using the polymerase chain reaction. Amplified products were analyzed for specificity by Southern blot hybridization with a radiolabeled DNA probe internal to the primer templates. The presence of CD4 molecules (i.e., receptors for cell binding of HIV) on intestinal epithelial cells was assessed using monoclonal antibodies and immunofluorescence labeling. Fantini et al. found that undifferentiated HT-29-D4 cells express a CD4-related antigen that lacks the epitope known to be involved in recognition of the HIV envelope binding protein referred to as gp120. Undifferentiated HT-29-D4 cells were infected by both HIV-1 and HIV-2 strains. However, the infection did not alter cell growth. Differentiated HT-29-D4 cells were also infected, either through the apical brush border or the basolateral membrane. Although the CD4-like surface molecules were restricted to the basolateral domain of differentiated cells, antibodies against specific epitopes of the CD4 glycoprotein did not inhibit HIV infection. The authors conclude that intestinal epithelial cells can be infected by both HIV-1 and HIV-2 via either the apical membrane or the basolateral membrane. There was no evidence of viral replication by reverse transcriptase activity. However, following culture of HT-29-D4 cells with virus, HIV DNA could be recovered by gene amplification as long as 9 months after exposure of intestinal tissue culture cells to the virus. The authors speculate that this might provide an explanation for the prolonged seronegative state observed in HIV-infected individuals. These findings raise the possibility that infected lamina propria lymphocytes and macrophages could coinfect intestinal epithelial cells. In addition, HIV present in the intestinal lumen could bind to the apical surface and infect enterocytes.

Galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type 1 on human colon epithelial HT29 cells

The gastrointestinal tract is considered to be a major route of infection for human immunodeficiency virus (HIV). Infection of human colon epithelial cells by HIV is not blocked by anti-CD4 antibodies known to block infection of lymphoid cells (J. Fantini, N. Yahi, and J. C. Chermann, Proc. Natl. Acad. Sci. USA 88:9297-9301, 1991), suggesting the presence of an alternate receptor for HIV on these cells. In this report, we show that (i) a monoclonal antibody specifically directed against galactosyl ceramide inhibited the infection of HT29 cells by two markedly different strains of HIV-1, as assessed by polymerase chain reaction amplification and reverse transcriptase assay; (ii) this antibody strongly labeled the surface of HT29 cells by immunofluorescence and electron microscopic immunolocalization; (iii) the labeling was preferentially but not totally restricted to the basolateral membrane domain of differentiated colonic cells, in agreement with the ability of HIV to infect both the apical and basolateral surfaces of these epithelial cells; and (iv) in thin-layer chromatography-immunostaining experiments with neutral glycolipids prepared from HT29 cells, the antibody specifically reacted with a ceramide monoglycoside fraction corresponding to galactosyl ceramide. We did not detect this glycolipid in lymphoid cells, and anti-galactosyl ceramide antibodies consistently failed to inhibit HIV infection of these cells. These data suggest that galactosyl ceramide (or a derivative) is an essential component of the receptor for HIV on the surface of HT29 cells.

Investigations into the mechanism by which sulfated polysaccharides inhibit HIV infection in vitro.

Sulfated polysaccharides have been shown to inhibit human immunodeficiency virus (HIV) infection in vitro. Dextrin sulfate, fucoidan, and dextran sulfate fail to neutralize virions directly, but interact with target cells to inhibit virus entry. Ionic interactions of sulfated polyanions with oppositely charged cell surface components, including CD4, have been assumed to be the inhibitory mechanism. It is shown that the sulfated polysaccharides inhibit infection of both CD4+ and CD4- cell lines by HIV and also that they inhibit HTLV-1 and, to a lesser extent, the simian retrovirus, MPMV, which use receptors other than CD4. One binding site for radiolabeled fucoidan on the surface of human T cells is an 18 kD protein, but its significance is not yet clear.

Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120.

Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41.

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C'' and FG. This structure is consistent with C'C'' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.

Inhibitory Effect of Tachyplesin I on the Proliferation of Human Immunodeficiency Virus in vitro

An antimicrobial peptide, tachyplesin I, isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus) was examined for its inhibitory effects on human immunodeficiency virus (HIV) infection in vitro. At a concentration of 7.5 micrograms/ml, tachyplesin I suppressed the development of cytopathic effects (CPE) by more than 70% in MT-4 cells infected with HIV (lymphadenopathy-associated virus). This inhibitory effect was observed only when the drug was added during the adsorption period of the virus to the cells. In cocultures of MOLT-4 and persistently HIV-infected cells (MOLT-4/HIV), tachyplesin I at the same concentration completely inhibited multinucleated giant cell formation. Infectivity of HIV was reduced by 10(-2.5) in medium free from fetal calf serum containing tachyplesin I at a concentration of 200 micrograms/ml. Tachyplesin I did not show any inhibitory effect on reverse transcriptase activity of HIV at concentrations of 9-80 micrograms/ml at which tachyplesin I inhibited HIV infection. These results suggest that the anti-HIV action of tachyplesin I was due to the inhibition of virus adsorption.

Effects of succinylated concanavalin A on infectivity and syncytial formation of human immunodeficiency virus

The effects of various lectins on the infectivity of human immunodeficiency virus (HIV) type 1 was investigated. Among the 25 lectins investigated, 2 types of concanavalin A (Con A) and 3 types of phytohemagglutinin were found to inhibit HIV infection. Succinylated Con A (S-Con A) efficiently blocked HIV-induced formation of syncytia in a coculture of MOLT-4 cells and blocked cell-free infection by HIV of MT-4 cells. The HIV-binding study revealed that S-Con A only partially inhibited viral binding to cells, although the control Leu-3a monoclonal antibody strongly inhibited it. When S-Con A was added to cultures after the initiation of viral adsorption, the number of HIV antigen-positive cells that developed depended on the time interval before addition of the compound. S-Con A inhibited HIV infection even after viral binding to cells at 0 degrees C and further incubation at 37 degrees C for 1 day. These data suggest that S-Con A inhibited mainly the fusion process rather than viral binding to cells in exerting its anti-HIV activity.