A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.     

Simian T-cell lymphotropic virus type 1 from Mandrillus sphinx as a simian counterpart of human T-cell lymphotropic virus type 1 subtype D

A recent serological and molecular survey of a semifree-ranging colony of mandrills (Mandrillus sphinx) living in Gabon, central Africa, indicated that 6 of 102 animals, all males, were infected with simian T-cell lymphotropic virus type 1 (STLV-1). These animals naturally live in the same forest area as do human inhabitants (mostly Pygmies) who are infected by the recently described human T-cell lymphotropic virus type 1 (HTLV-1) subtype D. We therefore investigated whether these mandrills were infected with an STLV-1 related to HTLV-1 subtype D. Nucleotide and/or amino acid sequence analyses of complete or partial long terminal repeat (LTR), env, and rex regions showed that HTLV-1 subtype D-specific mutations were found in three of four STLV-1-infected mandrills, while the remaining monkey was infected by a different STLV-1 subtype. Phylogenetic studies conducted on the LTR as well as on the env gp21 region showed that these three new STLV-1 strains from mandrills fall in the same monophyletic clade, supported by high bootstrap values, as do the sequences of HTLV-1 subtype D. These data show, for the first time, the presence of the same subtype of primate T-cell lymphotropic virus type 1 in humans and wild-caught monkeys originating from the same geographical area. This strongly supports the hypothesis that mandrills are the natural reservoir of HTLV-1 subtype D, although the possibility that another monkey species living in the same area could be the original reservoir of both human and mandrill viruses cannot be excluded. Due to the quasi-identity of both human and monkey viruses, interspecies transmission episodes leading to such a clade may have occurred recently.     

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.     

The V3-directed immune response in natural human immunodeficiency virus type 1 infection is predominantly directed against a variable, discontinuous epitope presented by the gp120 V3 domain

The specific binding of antibodies to the V3 loop in sera from human immunodeficiency type 1 (HIV-1)-infected individuals was investigated. Different V3 structures were analyzed as full-length loops or by pepscan. Our data show that on full-length V3 loops, both variable regions on either side of the tip of the loop (GPGRAF) contribute to a common epitope for type-specific antibodies. Type-specific antibodies bound strongly and at high titers to native V3 loops but negligibly once the loop was denatured. In contrast to the type-specific, discontinuous epitope, the linear, conserved epitopes presented by the full-length V3 loop, the tip, the amino-terminal base, and the carboxy-terminal base were not accessible to serum antibody. When the V3 sequences were analyzed with linear peptides, antibodies bound preferentially to peptides containing the conserved GPGRAF sequence. Thus, two different specificities of V3-directed antibodies were detected in patient sera. Unlike group-specific antibodies directed against GPGRAF peptides, lack of type-specific antibodies directed against the discontinuous epitope was correlated with viral escape from autologous neutralization. Our data suggest that the full-length conformation of the V3 loop is accessible predominantly to highly type-specific antibodies present in sera from HIV-1-infected individuals. These antibodies are directed against discontinuous V3 epitopes, not against conserved linear V3 targets. The implications of these findings for viral escape and blockade of infection with V3-based vaccines are discussed.     

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.     

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.     

Enhanced human T-cell lymphotropic virus type I expression following induction of the cellular stress response

Human T-cell lymphotropic virus type I (HTLV-I) infection is typically associated with long incubation periods between virus exposure and disease manifestation. Although viral protein expression is considered to play an important role in the pathogenesis of HTLV-I-associated diseases, limited information is known regarding host cell mechanisms that control viral gene expression. This study was designed to evaluate modulation of HTLV-I gene expression following induction of the cellular stress response in HTLV-I-infected lymphocytes. The cellular stress response was elicited by treatment with either Na arsenite or thermal stress and was monitored by demonstrating increased expression of the 72-kDa heat shock protein. Induction of the cellular stress response in HTLV-I-infected lymphocytes resulted in significantly increased HTLV-I-mediated syncytia formation due to enhanced HTLV-I envelope (gp46) expression. Intracellular viral proteins and released p24 capsid protein were increased in stressed infected lymphocytes as compared to nonstressed infected lymphocytes. Furthermore, HTLV-I-LTR reporter gene constructs had increased activity (three- to sixfold) in a transiently transfected, uninfected lymphocyte cell line following induction of the cellular stress response. Quantitation of HTLV-I RNA expression by slot blot analysis of infected lymphocytes suggested variable increases in RNA accumulation. Northern blot analysis demonstrated no qualitative changes in expression of RNA species. These data suggest a relationship between modulation of viral replication and a basic cellular response to stress and have important implications for understanding host cell control mechanisms of HTLV-I expression.     

CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

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.     

Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant Fab fragments

Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 microg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 microg/mL.     

The quantitative humoral immune response to the hepatitis C virus is correlated with disease activity and response to interferon-alpha

BACKGROUND/AIM: Virus-host interactions may have pathogenetic significance in chronic hepatitis. Thus the humoral immune response was evaluated during the clinical course of HCV-infected patients. METHODS: Eighteen selected chronic HCV patients received three doses of 3 or 6 MU interferon-alpha 2a weekly for 6 to 12 months and were followed up for 6 to 60 months. Anti-HCV antibody levels were serially measured either in end-point diluted sera with the Matrix-Assay or with quantitative anti-HC34-IgG and -IgM ELISA. Circulating immune complexes were assessed by flow cytometry and the results were correlated with histology, quantitative HCV-RNA levels and genotypes. RESULTS: Nine complete responders (CR; genotypes 1a n = 4; 1b n = 1; 2a n = 1; 3a n = 3) showing sustained virus elimination and ALT normalisation had low HCV-RNA pretreatment levels (mean 14 x 10(3) copies/ml) compared to six nonresponders and three partial responders (NR/PR; genotypes 1a n = 2; 1b n = 7) who had significantly higher HCV-RNA pretreatment levels (mean 254 x 10(3) copies/ml; p < 0.01). In untreated NR/PR the HC34 core-antigen was most immunogenic, in CR the NS3-derived HC29-antigen. Pre-treatment levels of anti-HC 34-IgG and -IgM antibody levels in NR/PR were higher than in CR (IgM/IgG p = 0.05, n.s.) and these differences became significant during or after therapy (3 months therapy: IgM p < 0.02/IgG p < 0.07; end of therapy: IgM 0.006/IgG p < 0.04; 6 months post-therapy: IgM p < 0.002/IgG p < 0.004). The PR patients showed recurrent anti-HC34 antibody levels that preceded disease reactivation and detectable HCV-RNA in serum. Immune complex formation increased in some patients during treatment but did not correlate with disease activity, quantitative viraemia, antibody levels or therapy outcome. CONCLUSION: Anti-HC34 antibodies, i.e. of the IgM-subtype, correlated quantitatively with viraemia and disease activity. Monitoring the antibody levels may predict the long-term therapy outcome during interferon-alpha treatment.     

Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.     

Induction of tumor necrosis factor alpha in human neuronal cells by extracellular human T-cell lymphotropic virus type 1 Tax

To examine the role of human T-lymphotropic virus type 1 (HTLV-1) Tax1 in the development of neurological disease, we studied the effects of extracellular Tax1 on gene expression in NT2-N cells, postmitotic cells that share morphologic, phenotypic, and functional features with mature human primary neurons. Treatment with soluble HTLV-1 Tax1 resulted in the induction of tumor necrosis factor alpha (TNF-alpha) gene expression, as detected by reverse-transcribed PCR and by enzyme-linked immunosorbent assay. TNF-alpha induction was completely blocked by clearance with anti-Tax1 monoclonal antibodies. Furthermore, cells treated with either a mock bacterial extract or with lipopolysaccharide produced no detectable TNF-alpha. Synthesis of TNF-alpha in response to soluble Tax1 occurred in a dose-dependent fashion between 0.25 and 75 nM and peaked within 6 h of treatment. Interestingly, culturing NT2-N cells in the presence of soluble Tax1 for as little as 5 min was sufficient to result in TNF-alpha production, indicating that the induction of TNF-alpha in NT2-N does not require Tax1 to be continually present in the culture medium. Treatment of the undifferentiated parental embryonal carcinoma cell line NT2 with soluble Tax1 did not result in TNF-alpha synthesis, suggesting that differentiation-dependent, neuron-specific factors may be required. These results provide the first experimental evidence that neuronal cells are sensitive to HTLV-1 Tax1 as an extracellular cytokine, with a potential role in the pathology of HTLV-1-associated/tropical spastic paraparesis.     

Neutralization of human immunodeficiency virus type 1 by antibody to gp120 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity

We investigated the relative importance of binding site occupancy and epitope specificity in antibody neutralization of human immunodeficiency virus (HIV) type 1 (HIV-1). The neutralization of a T-cell-line-adapted HIV-1 isolate (MN) was analyzed with a number of monovalent recombinant Fab fragments (Fabs) and monoclonal antibodies with a range of specificities covering all confirmed gp120-specific neutralization epitopes. Binding of Fabs to recombinant monomeric gp120 was determined by surface plasmon resonance, and binding of Fabs and whole antibodies to functional oligomeric gp120 was determined by indirect immunofluorescence and flow cytometry on HIV-infected cells. An excellent correlation between neutralization and oligomeric gp120 binding was observed, and a lack of correlation with monomeric gp120 binding was confirmed. A similar degree of correlation was observed between oligomeric gp120 binding and neutralization with a T-cell-line-adapted HIV-1 molecular clone (Hx10). The ratios of oligomer binding/neutralization titer fell, in general, within a relatively narrow range for antibodies to different neutralization epitopes. These results suggest that the occupancy of binding sites on HIV-1 virions is the major factor in determining neutralization, irrespective of epitope specificity. Models to account for these observations are proposed.     

Transmission of human T-cell leukemia virus type 1 to mice

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10(5) spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.     

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.     

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.