Coexpression of Human Immunodeficiency Virus Envelope Proteins and Tat from a Single Simian Virus 40 Late Replacement Vector (original) (raw)
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Murine retroviral vector that induces long-term expression of HIV-1 envelope protein
Journal of Virological Methods, 1994
A retroviral vector was constructed that induces long-term expression of human immunodeficiency virus type 1 (HIV-l) reu, vpu and env genes. The vector contains the neo gene and a cytomegalovirus (CMV) immediate early promoter followed by HIV-l sequence. When HeLa cells were infected with viral stocks derived from this vector, about 25% of the resulting G418-resistant clones expressed HIV-l envelope protein (Env), easily detectable by Western blot analysis, metabolic labelling, and syncytium formation after co-cultivation with HeLa-CD4 cells. In most cases the level of Env expression was higher than in a T cell line (H9) chronically infected with HIV-l. Env-expressing HeLa cell lines also expressed Rev, detected by transfection with a Rev-dependent CAT gene construct, and Vpu, detected by immunoprecipitation with a Vpu-specific antiserum. The 75% of G418-resistant HeLa cell lines that did not express Env were found to contain proviruses that had undergone deletion of env sequences corresponding to a known intron; presumably these cell lines arose as a result of infection with virions derived from spliced RNAs. This vector should be useful for studying non-transient effects of HIV Env, Rev and Vpu in tissue culture, and for the production of Env-and/or Rev-expressing cell lines.
Journal of virology, 1989
A recombinant adenovirus was constructed by inserting the human immunodeficiency virus type 1 (HIV-1) envelope gene downstream from the early region 3 (E3) promoter of adenovirus type 5 (Ad5), replacing the coding sequences of E3. The recombinant virus replicated as efficiently as the parent virus in all cell lines tested. Human cells infected with the recombinant virus synthesized the HIV-1 envelope precursor gp160, which was efficiently processed to the envelope glycoproteins gp120 and gp41. A human T-lymphoblast line (Molt-4) infected with the recombinant virus expressed HIV-1 envelope glycoproteins on the cell surface, leading to syncytium formation. The envelope gene was expressed from the E3 promoter at early times after infection and at late times from the major late promoter. When cotton rats were infected with the recombinant virus, antibodies against the HIV-1 envelope glycoproteins could be expressed in an immunoreactive form by the recombinant adenovirus, further illustr...
Nature of Nonfunctional Envelope Proteins on the Surface of Human Immunodeficiency Virus Type 1
Journal of Virology, 2006
Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.
Traffic, 2000
proteins were monitored in a Mamu-A*01-positive rhesus macaque infected with SIVsmE660. Peripheral blood mononuclear cells (PBMC) cultured with synthetic peptides spanning the entire gp160 and Gag coding region recognized a total of three epitopes. One located in Gag was identified as the previously described Mamu-A*01-restricted p11cC3M epitope (CTPYDINQM). The other two epitopes, designated p15m and p54m, were located in the gp160 envelope protein. Both were nine amino acids in length and were predicted to bind Mamu-A*01 because they contained proline and leucine residues at positions 3 and 9, respectively. Indeed, expression of this class I major histocompatibility complex molecule was required for target cell recognition by envelope-specific CD8 ؉ T cells directed against both epitopes. These Mamu-A*01-restricted epitopes in the SIV envelope will be useful for monitoring immune responses in vaccinated or infected animals.
Journal of Virology, 1995
68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells.
Characterization of an HIV-1 point mutant blocked in envelope glycoprotein cleavage
Virology, 1990
The envelope proteins of retroviruses are derived from a polypeptide precursor protein by cleavage adjacent to a cluster of basic amino acids. Site-specific mutagenesis was used to construct a mutant of the human immunodeficiency virus type 1 (HIV-l) in which the arginine residue at the carboxy-terminus of the gpl20 was changed to a threonine residue. This single substitution was sufficient to abolish all detectable cleavage of the gp160 envelope precursor polypeptide as well as virus infectivity. The gpl60 was produced in normal quantities from a biologically active clone of the mutant virus after transfection into cos-1 cells. The mutant gpl60 contained N-linked oligosaccharide chains with mannose-rich cores similar to those of the gp160 produced by the wild-type clone. lmmunofluorescence assays showed that gp160 was transported to the surface of transfected CD4+ HeLa cells. No envelope proteins of known size could be detected in the media of cells transfected with the mutant virus, suggesting that functional virions were not formed. Binding of the mutant gp160 to the CD4 receptor molecule was unimpaired. Despite this and the presence of gpl60 on the cell surface, neither growth of mutant-transfected CD4+ HeLa cells nor cocultivation of transfected cos-1 cells with H9 cells resulted in significant syncytium formation. The data indicate that the carboxy-terminal arginine residue of HIV-1 gpl20 is necessary for envelope protein cleavage and suggest cleavage is important in the virus life cycle in both functional virus release and membrane fusion. o 1990 Academic PESS. I~C.
Expression of HIV-1 envelope gene by recombinant avipox viruses
Vaccine, 1994
Recombinant canarypox (CP) and fowlpox (FP) viruses that contained two forms of the HIV-1 (SF2 strain) env 9ene were engineered and their expression analysed in chick, simian and human cells. These vectors can efficiently replicate in avian but not in mammalian cells, in which infection is abortive. The two forms, consistin9 of the entire env open reading frame (IS +) or of the same 9ene lackin9 the putative immunosuppressive (IS-) region (amino acids 583-599), were individually inserted into the two virus vector backgrounds. In order to avoid premature transcription termination of the foreign 9ene and to improve protein expression, a mutagenesis was also performed within the T5NT motif without alterin9 the amino acid sequence. By immunoprecipitation analyses, cells infected with CP and FP recombinants expressed HIV-1 env polypeptides of the appropriate molecular weight. We observed that the 91)160 precursor was proteolytically cleaved except in MRC-5 cells infected with the IS-recombinants and that these polypeptides were 9lycosylated. Further analysis of these recombinant viruses by indirect immunofluorescence and syncytia inhibition assays indicated that the 9p120/gp41 complex was present on the surface of infected cells, the number of syncytia bein9 significantly lower when cells were infected by the CPIS-or FPIS-recombinants. Moreover, sera of immunized rabbits revealed the presence oJspecific antibodies in animals inoculated either with CP or with FP recombinants. These new constructs, which are unable to support a productive infection in human cells', might therefore also be a 9ood anti-HIV-1 candidate vaccine in seropositive hosts.
Journal of Virology, 1989
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-c...