Apoptosis induced in CD4+ cells expressing gp160 of human immunodeficiency virus type 1.

In a previous study (Y. Koga, M. Sasaki, H. Yoshida, H. Wigzell, G. Kimura, and K. Nomoto, J. Immunol. 144:94-102, 1990), we demonstrated that the expression of gp160, a precursor form of envelope glycoprotein of human immunodeficiency virus type 1, in CD4+ cells causes the downregulation of surface CD4 and single-cell killing by forming intracellular gp160-CD4 complex. In the present study we investigated the events that lead to cell death in CD4+ cells expressing gp160. We found that apoptosis is induced in cells undergoing single-cell death. Moreover, even the cell clone, which expresses so little gp160 that it does not exhibit any apparent cytopathic effects, such as the inhibition of cell growth, was found to be highly susceptible to the apoptosis induction by the anti-Fas monoclonal antibody.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160.

In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens.     

Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing

The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline). Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 μM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.     

Polyarginine inhibits gp160 processing by furin and suppresses productive human immunodeficiency virus type 1 infection

Correct endoproteolytic maturation of gp160 is essential for the infectivity of human immunodeficiency virus type 1. This processing of human immunodeficiency virus-1 envelope protein, gp160, into gp120 and gp41 has been attributed to the activity of the cellular subtilisin-like proprotein convertase furin. The prototypic furin recognition cleavage site is Arg-X-Arg/Lys-Arg. Arg-Arg-Arg-Arg-Arg-Arg or longer iterations of polyarginine have been shown to be competitive inhibitors of substrate cleavage by furin. Here, we tested polyarginine for inhibition of productive human immunodeficiency virus-1-infection in T-cell lines, primary peripheral blood mononuclear cells, and macrophages. We found that polyarginine inhibited significantly human immunodeficiency virus-1 replication at concentrations that were benign to cell cultures ex vivo and mice in vivo. Using a fluorogenic assay, we demonstrated that polyarginine potently inhibited substrate-specific proteolytic cleavage by furin. Moreover, we verified that authentic processing of human immunodeficiency virus-1 gp160 synthesized in human cells from an infectious human immunodeficiency virus-1 (HIV-1) molecular clone was effectively blocked by polyarginine. Taken together, our data support that inhibitors of proteolytic processing of gp160 may be useful for combating human immunodeficiency virus-1 and that polyarginine represents a lead example of such inhibitors.     

Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.

The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.     

A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1.

Furin, a subtilisin-like mammalian endoprotease, is thought to be responsible for the processing of many proprotein precursors of cellular and viral origin, including gp160 of human immunodeficiency virus type 1, which share the consensus processing site motif, Arg-X-Lys/Arg-Arg, for protease recognition (for reviews, see P. J. Barr, Cell 66:1-3, 1991, and Y. Nagai, Trends Microbiol. 1:81-87, 1993). To confirm and extend the concept that gp160 is processed by furin, we used here a cell line, LoVo, which was recently demonstrated to be furin defective. Unexpectedly, LoVo cells were found to process gp160 as efficiently as normal cell lines do, hence being able to fuse with CD4-expressing HeLa cells and to produce fully infectious virions. On the other hand, the same cell line was almost totally incapable of processing Newcastle disease virus fusion glycoprotein with a similar oligobasic cleavage recognition motif, providing a strong case for furin-mediated processing. Our present study thus raises a further need to search for and identify the proteinases involved in human immunodeficiency virus type 1 gp160 processing rather than supporting the notion that furin is responsible.     

Processing of the envelope glycoprotein gp160 in immunotoxin-resistant cell lines chronically infected with human immunodeficiency virus type 1.

We describe the isolation and characterization of variant cell lines which are chronically infected with the human immunodeficiency virus (HIV) and resistant to the action of immunotoxins directed against the HIV envelope protein. These variants all produce normal levels of HIV proteins, budding virions, and the envelope protein precursor gp160. Two of the variants, 10E and 11E, contain a mutation within the env gene which results in the production of a truncated precursor and altered processing and transport of the protein to the cell surface. Variants B9 and G4 are defective in gp160 cleavage and do not efficiently transport the envelope protein to the cell surface. There are no mutations in the expressed viruses of B9 and G4. These cell lines express higher levels of CD4 protein and mRNA than H9/NL4-3. Thus, 10E, 11E, B9, and G4 have escaped immunotoxin action by downmodulating the envelope protein from their cell surfaces. None of these variants produce infectious HIV. Two other immunotoxin-resistant variants, E9-3 and 41-17, produce normal levels of gp160, efficiently transport the cleaved and processed subunits to the cell surface, and secrete infectious HIV. These studies identify alterations in gp160 processing that underscore the importance of the relationship between HIV and the cell that it infects.     

Topogenic analysis of the human immunodeficiency virus type 1 envelope glycoprotein, gp160, in microsomal membranes

The orientation in cellular membranes of the 856 amino acid envelope glycoprotein precursor, gp160, of human immunodeficiency virus type 1 was investigated in vitro. Variants of the env gene were transcribed using the bacteriophage SP6 promoter, translated using a rabbit reticulocyte lysate, and translocated into canine pancreatic microsomal membranes. Immunoprecipitation studies of gp160 variants using antibodies specific for various gp160-derived polypeptides provided evidence that the external (cell surface) domain of gp160 begins at the mature amino terminus of the protein and continues through amino acid 665. A stop-transfer sequence (transmembrane domain) was identified in a hydrophobic region COOH-terminal to amino acid 665 and NH2-terminal to amino acid 732. Protease protection experiments demonstrated that gp160 possesses a single cytoplasmic domain COOH-terminal to residue 707. Membrane extraction studies using carbonate buffer provided evidence that the 29 amino acid hydrophobic domain (residues 512-541) of gp160 was unable to serve as a stop-transfer sequence. Finally, we propose that the cytoplasmic tail of gp160 forms a secondary association with the microsomal membranes.     

Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160.

The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.     

A mechanism of restricted human immunodeficiency virus type 1 expression in human glial cells.

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.     

Inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides

As the limitations of antiretroviral drug therapy, such as toxicity and resistance, become evident, interest in alternative therapeutic approaches for human immunodeficiency virus (HIV) infection is growing. We developed the first gene therapeutic strategy targeting entry of a broad range of HIV type 1 (HIV-1) variants. Infection was inhibited at the level of membrane fusion by retroviral expression of a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 gp41 transmembrane glycoprotein. To achieve maximal expression and antiviral activity, the peptide itself, the scaffold for presentation of the peptide on the cell surface, and the retroviral vector backbone were optimized. This optimized construct effectively inhibited virus replication in cell lines and primary blood lymphocytes. The membrane-anchored C-peptide was also shown to bind to free gp41 N peptides, suggesting that membrane-anchored antiviral C peptides have a mode of action similar to that of free gp41 C peptides. Preclinical toxicity and efficacy studies of this antiviral vector have been completed, and clinical trials are in preparation.     

Inhibition of gp160 and CD4 maturation in U937 cells after both defective and productive infections by human immunodeficiency virus type 1.

Our results demonstrate that the formation of intracellular complexes between the envelope glycoprotein precursor gp160 of human immunodeficiency virus type 1 and CD4 is a major event, leading to the disappearance of CD4 at the cell surface of infected U937 cells. Using both productively and defectively infected clones of U937 cells, we assessed the effect of CD4-gp160 intracellular association on the maturation of both proteins. Pulse-chase labeling followed by sequential immunoprecipitation was used to analyze the processing of both free and associated CD4 and gp160, and the results showed that the trimming, proteolytic cleavage, and degradation of gp160 were completely abrogated after intracellular binding to CD4. Similarly, the maturation process which normally transforms 80% of CD4 to a partially endoglycosidase H-resistant species was also impaired subsequent to the formation of these complexes. A comparison of gp160 maturation either in free form or as a CD4 complex revealed that neither inefficient transport nor degradation of gp160 can account for the observed blockage of CD4 maturation. Moreover, this impairment was independent of gp120 and gp41, since a defective clone of human immunodeficiency virus type 1-infected cells, unable to cleave gp160, showed binding of CD4 and inhibition of CD4 transport and maturation with the same efficiency as occurred in productively infected cells. Expression of gp160 is thus necessary and sufficient to cause CD4 receptor down-modulation for both productively and defectively infected cells.     

The carboxy terminus of human immunodeficiency virus type 1 gp160 limits its proteolytic processing and transport in transfected cell lines.

Mutagenesis of the transmembrane domain and cytoplasmic tail of human immunodeficiency virus type 1 envelope glycoprotein gp160 revealed that its intracellular transport and processing in transfected cell lines were modulated by a functional domain included in the carboxy-terminal sequence consisting of residues 751 to 856.     

Expression of membrane-associated and secreted variants of gp160 of human immunodeficiency virus type 1 in vitro and in continuous cell lines.

The gene encoding the 856-amino-acid envelope glycoprotein, gp160, of human immunodeficiency virus type 1 was mutagenized and transfected into Chinese hamster ovary cells. Continuous cell lines that constitutively produced gp160 variants were isolated and used to study the biosynthesis and orientation of gp160 in cellular membranes. In vivo studies of gp160 variants failed to reveal domains upstream of amino acid residue 665 that could serve as stop transfer sequences. Analyses of gp160 variants expressed in vitro in a translation-coupled translocation system were consistent with the in vivo studies and provided evidence that gp160 is a simple bitopic membrane protein. A model for the orientation and function of gp160 in cellular membranes is presented. The cell lines described provide a convenient source of the gp120-gp41 complex.     

Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gp160, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gp160 into the mature virion components, gp120 and gp41. We found that coexpression of gp160 and human CD4 in HeLa cells severely impaired gp120 production due to the formation of intracellular gp160-CD4 complexes. This CD4-mediated inhibition of gp160 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gp160 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gp160-CD4 complexes.     

Model for intracellular folding of the human immunodeficiency virus type 1 gp120.

The intracellular folding of the human immunodeficiency virus type 1 gp120 has been assessed by analyzing the ability of the glycoprotein to bind to the viral receptor CD4. Pulse-chase experiments revealed that the glycoprotein was initially produced in a conformation that was unable to bind to CD4 and that the protein attained the appropriate tertiary structure for binding with a half-life of approximately 30 min. The protein appears to fold within the rough endoplasmic reticulum, since blocking of transport to the Golgi apparatus by the oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone did not appear to perturb the folding kinetics of the molecule. The relatively lengthy folding time was not due to modification of the large number of N-linked glycosylation sites on gp120, since inhibition of the first steps in oligosaccharide modification by the inhibitors deoxynojirimycin or deoxymannojirimycin did not impair the CD4-binding activity of the glycoprotein. However, production of the glycoprotein in the presence of tunicamycin and removal of the N-linked sugars by endoglycosidase H treatment both resulted in deglycosylated proteins that were unable to bind to CD4, suggesting in agreement with previous results, that glycosylation contributes to the ability of gp120 to bind to CD4. Interestingly, incomplete endoglycosidase H treatment revealed that a partially glycosylated glycoprotein could bind to the receptor, implying that a subset of glycosylation sites, perhaps some of those conserved in different isolates of human immunodeficiency virus type 1, might be important for binding of the viral glycoprotein to the CD4 receptor.     

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1.

Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.     

Disturbance of nuclear transport of proteins in CD4+ cells expressing gp160 of human immunodeficiency virus.

An extensive cytopathic effect occurred in the CD4+ cells expressing gp160 of human immunodeficiency virus. A protein capable of nuclear location that was microinjected into such cells was not transported into the nuclei at an early stage when little cytopathic effect had yet to occur.