Human submandibular saliva inhibits human immunodeficiency virus type 1 infection by displacing envelope glycoprotein gp120 from the virus

Actin is a highly conserved, ubiquitous cytoskeletal protein, which is essential for multiple cellular functions. Despite its small size (Mr = 42 000), unpolymerized forms of actin, as well as polymerized forms, exist primarily in the cytoplasm, excluded from the nucleus. Although spatial control of actin is crucially important, the molecular mechanisms ensuring the cytoplasmic localization of unpolymerized actin have not been revealed so far. In this paper we report that actin contains two leucine-rich type nuclear export signal (NES) sequences in the middle part of the molecule, which are both shown to be functional. Monomeric actin, when injected into the nucleus, was rapidly exported in a manner which was sensitive to leptomycin B (LMB), a specific inhibitor of NES-dependent nuclear export. LMB treatment of cells prevented nuclear exclusion of endogenous actin, inducing its nuclear accumulation. Furthermore, actin mutants with disrupted NESs accumulated in the nucleus. Expression of these NES-disrupted actin mutants, but not of wild-type actin, induced a decrease in the proliferative potential of the cell. These results reveal a novel molecular mechanism controlling the subcellular distribution of actin.     

Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.     

Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef

nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Molecular cloning and characterization of viruses isolated from chimpanzees with pathogenic human immunodeficiency virus type 1 infections

We have previously described the development of AIDS in a chimpanzee (C499) infected with human immunodeficiency virus type 1 (HIV-1) and the subsequent pathogenic HIV-1 infection in another chimpanzee (C455) transfused with blood from C499 (F. J. Novembre et al., J. Virol. 71:4086-4091, 1997). In the present study, two virus isolates were derived from these animals: HIV-1JC from peripheral blood mononuclear cells (PBMC) of C499, and HIV-1NC from plasma of C455. These virus isolates were used to generate two infectious molecular clones, termed HIV-1JC16 and HIV-1NC7 (JC16 and NC7, respectively). Comparative analyses of the sequences of the two clones showed that they were highly interrelated but distinct. Based on heteroduplex mobility assays, JC16 and NC7 appear to represent dominant viruses in the uncloned stock population. Compared with amino acid sequences of the parental viruses HIV-1SF2, HIV-1LAV-1b, and HIV-1NDK, JC16 and NC7 showed a number of differences, including insertions, deletions, and point mutations spread throughout the genome. However, insertion/deletion footprints in several genes of both JC16 and NC7 suggested that recombination between SF2 and LAV-1b could have occurred, possibly contributing to the generation of a pathogenic virus. Comparative in vitro analyses of the molecular clones and the uncloned stocks of HIV-1JC and HIV-1NC revealed that these viruses had strikingly similar replicative abilities in mitogen-stimulated PBMC and in macrophages. Compared to the SF2 and LAV-1b isolates of HIV-1, HIV-1JC and HIV-1NC isolates were more similar to LAV-1b with respect to the ability to replicate in mitogen-stimulated PBMC and macrophages. These viruses should prove to be useful in mapping determinants of pathogenesis.     

Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design

Understanding the antibody response in HIV-1 infection is important to vaccine design. We have studied the antibody response to HIV-1 envelope at the molecular level and determined the characteristics of neutralizing and non-neutralizing antibodies. These antibodies were isolated from phage display libraries prepared from long-term seropositive asymptomatic individuals. The HIV-1 envelope is presented to the immune system in several antigenically distinct configurations: unprocessed gp160, gp120 and gp41 subunits and native envelope, each of which may be important in eliciting an antibody response in HIV-1 infection. The antibodies tested characteristically had poor affinities for native envelope as expressed on the surface of virions or infected cells, but had high affinities against non-native forms of HIV-1 envelope (viral debris). An exceptionally potent neutralizing antibody in contrast, bound native envelope with equivalent or somewhat higher affinity than this. This indicates that the antibody response in HIV-1 infection is principally elicited by viral debris rather than virions, and that these antibodies bind and neutralize viruses sub-optimally. Potential vaccines should be designed to elicit responses against native envelope.     

Interaction of human immunodeficiency virus type 1 envelope glycoprotein V3 loop with CCR5 and CD4 at the membrane of human primary macrophages

We show that infection of primary monocyte-derived macrophages (MDMs) and blood lymphocytes (PBLs) by human immunodeficiency virus type 1 (HIV-1) R5 strains, but not that of PBLs by X4 strain HIV-1LAI, is inhibited by beta-chemokines RANTES and MIP-1alpha. A biotinylated disulfide-bridged peptide mimicking the complete loop of clade B consensus V3 domain of gp120 (V3Cs), but not a biotinylated V3LAI peptide or a control beta-endorphin peptide of approximately the same molecular weight (MW), was found to bind specifically to MDM membrane proteins, in particular two proteins of 42 and 62 kDa migrating as sharp bands after electroblotting onto Immobilon, and this was specifically inhibited by anti-V3 antibodies. When biotinylated V3Cs was incubated with intact MDMs, which were then washed and lysed, and the resulting material was incubated with streptavidin-agarose beads and electroblotted onto Immobilon, fresh V3Cs also bound to proteins of the same molecular weight recovered in the V3Cs-interacting material. This binding was inhibited by anti-V3 antibodies, and no binding occurred with the control peptides. V3Cs also bound to soluble recombinant CD4, and CD4 monoclonal antibody Q4120 specifically recognized the V3Cs-interacting 62-kDa protein, which should thus correspond to CD4. Recombinant radiolabeled RANTES, MIP-1alpha, and MIP-1beta, but not IL-8, also bound to a 42-kDa protein on the membrane of MDMs as well as to the V3Cs-interacting 42-kDa protein, and excess unlabeled V3Cs inhibited such binding. This protein was also recognized by antibodies to CCR5, the RANTES/MIP-1alpha/MIP-1beta receptor. These data show that V3Cs binds to MDM membrane proteins that comprise CD4 and CCR5, and that multimolecular complexes involving at least gp120 V3, CD4, and CCR5 are formed on the surface of MDMs as part of V3-mediated postbinding events occurring during HIV-1 infection.     

An investigation of the high-avidity antibody response to glycoprotein 120 of human immunodeficiency virus type 1

The avidity of antibodies for antigens can be measured by determining what remains bound after exposing the antibody-antigen complex to a chaotropic agent such as urea. This method has been gaining popularity for assessing the immune response to the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120 (or its counterpart from simian immunodeficiency virus), during natural infection or after subunit vaccination. High-avidity antibodies have been considered to be a possible correlate of protection. We have examined the avidity assay to determine what it, in fact, measures. First, we studied the development of the anti-gp120 response in seroconverting individuals. Urea elution reduced the polyclonal anti-gp120 titers by 3- to 10-fold. After allowing for the consequent reduction in assay sensitivity, there was no obvious change in the rate of development of the high-avidity and unfractionated antibody responses. Furthermore, in the one individual who developed a strong autologous, virus-neutralizing response, the appearance of neutralizing antibodies and high-avidity antibodies did not coincide. Antibodies to the V3 loop, when present, comprised a major fraction of the polyclonal response that survives urea elution. We next examined the effect of urea elution on the binding to gp120 of a panel of monoclonal antibodies (MAbs). Urea treatment preferentially eluted MAbs to discontinuous rather than continuous epitopes, independent of their affinities. Furthermore, these patterns of epitope stability were unaltered by the presence of polyclonal anti-gp120 antibodies. As most broadly neutralizing anti-gp120 antibodies recognize discontinuous epitopes, this skewing effect must be taken into account when interpreting studies using polyclonal sera.     

Truncation of the human immunodeficiency virus type 1 envelope glycoprotein allows efficient pseudotyping of Moloney murine leukemia virus particles and gene transfer into CD4+ cells

Human immunodeficiency virus type 1 (HIV-1) can readily accept envelope (Env) glycoproteins from distantly related retroviruses. However, we previously showed that the HIV-1 Env glycoprotein complex is excluded even from particles formed by the Gag proteins of another lentivirus, visna virus, unless the matrix domain of the visna virus Gag polyprotein is replaced by that of HIV-1. We also showed that the integrity of the HIV-1 matrix domain is critical for the incorporation of wild-type HIV-1 Env protein but not for the incorporation of a truncated form which lacks the 144 C-terminal amino acids of the cytoplasmic domain of the transmembrane glycoprotein. We report here that the C-terminal truncation of the transmembrane glycoprotein also allows the efficient incorporation of HIV-1 Env proteins into viral particles formed by the Gag proteins of the widely divergent Moloney murine leukemia virus (Mo-MLV). Additionally, pseudotyping of a Mo-MLV-based vector with the truncated rather than the full-length HIV-1 Env allowed efficient transduction of human CD4+ cells. These results establish that Mo-MLV-based vectors can be used to target cells susceptible to infection by HIV-1.     

Mutational analysis of residues in the coiled-coil domain of human immunodeficiency virus type 1 transmembrane protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263-273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426-430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

Changes in human immunodeficiency virus type 1 envelope glycoproteins responsible for the pathogenicity of a multiply passaged simian-human immunodeficiency virus (SHIV-HXBc2)

In vivo passage of a poorly replicating, nonpathogenic simian-human immunodeficiency virus (SHIV-HXBc2) generated an efficiently replicating virus, KU-1, that caused rapid CD4(+) T-lymphocyte depletion and AIDS-like illness in monkeys (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L.-J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996). The env gene of the KU-1 virus was used to create a molecularly cloned virus, SHIV-HXBc2P 3.2, that differed from a nonpathogenic SHIV-HXBc2 virus in only 12 envelope glycoprotein residues. SHIV-HXBc2P 3.2 replicated efficiently and caused rapid and persistent CD4(+) T-lymphocyte depletion in inoculated rhesus macaques. Compared with the envelope glycoproteins of the parental SHIV-HXBc2, the SHIV-HXBc2P 3.2 envelope glycoproteins supported more efficient infection of rhesus monkey peripheral blood mononuclear cells. Both the parental SHIV-HXBc2 and the pathogenic SHIV-HXBc2P 3.2 used CXCR4 but none of the other seven transmembrane segment receptors tested as a second receptor. Compared with the parental virus, viruses with the SHIV-HXBc2P 3.2 envelope glycoproteins were more resistant to neutralization by soluble CD4 and antibodies. Thus, changes in the envelope glycoproteins account for the ability of the passaged virus to deplete CD4(+) T lymphocytes rapidly and specify increased replicative capacity and resistance to neutralization.     

Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies

A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein.