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PubMed2004

[Study on the immunogenicity of HIV-1 gag vaccine].

AIM: To detect the immunogenicity of HIV-1 gag DNA vaccine. METHODS: The serum antibody titers of the immunized mice were detected by ELISA. The numbers of spleen's CD4(+) and CD8(+) T cells were analyzed by the fluorescent antibody staining and the cytotoxic activity was determined by lactate dehydrogenase (LDH) release assay. RESULTS: The titers of the serum antibodies and the numbers and cytotoxic activity of spleen's T lymphocyte subsets had significant differences (P<0.05 and P<0.01, respectively) between pVAXGAG and pVAX1 vaccinated groups. CONCLUSION: HIV-1 DNA vaccine plasmid pVAXGAG could elicit both the specific humoral and cellular immunities in BALB/c mice.

AIDS Vaccines↗
PubMed1992

[IgG autoantibodies against cellular p72 antigen crossing with (MLV) p15-gag antigen: presence in early HIV 1 infection, in HBV infection and in primary Gougerot-Sjögren].

IgG antibodies of autoimmune SLE (Systemic Lupus Erythematosus) serum S detected a HeLa hnRNP 72 kDa protein, cross-reacting with the retroviral (MLV) p15-gag polypeptide. Since serum S disclosed a ubiquitous 72 kDa antigen in HeLa cell fractions, was prepared the so-called cytoplasmic "X fraction", enriched for the 72 kDa protein, defined here as p72. This autoantigen was detected by antibodies of HIV 1+ patients, recently of seroconverted (RSC) asymptomatic subjects, of HBV+ sera, and of primary Gougerot-Sjögren (prGS) sera. The presence of these autoantibodies in different autoimmune and infectious pathologies raises the question of the involvement of p72 in the immune processes and in the early HIV1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Transforming potential of truncated v-myb and stimulation of replication by gag-myb fusion products.

We have previously reported that truncated forms of the v-myb oncogene of avian myeloblastosis virus (AMV) are expressed in transformed chicken embryo fibroblasts (CEF). In this paper, we show that deletion mutants encoding v-myb products altered in either the DNA-binding or the negative regulatory domains are able to induce CEF transformation. In addition, we report that recombinant plasmids expressing gag-myb fusion proteins are maintained as extrachromosomal forms in transfected cells. This observation provides an important clue for a possible role of myb in the DNA replication processes.

Animals↗
PubMed1991

Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys.

Infection of macaque monkeys with the simian immunodeficiency virus of macaques (SIVmac) results in disease similar to human AIDS. Therefore, the macaque monkey is proving to be an important model for testing the effectiveness of various AIDS vaccine approaches. A detailed analysis of the cellular immune responses is necessary for the evaluation of candidate vaccines. However, this has not been possible in macaques, due, in part, to the unknown nature of the MHC molecules that restrict their T lymphocytes. In our report we demonstrate that a particular MHC class I molecule involved in the rhesus monkey's effector T lymphocyte response to SIVmac is expressed at a high frequency in a colony of rhesus monkeys. SIVmac-infected monkeys that express this MHC class I molecule all develop CTL that are restricted by that molecule and recognize an identical nine amino acid epitope of the SIVmac gag protein. This MHC class I molecule has been defined as an HLA-A homolog by cDNA cloning and sequencing. It has also been expressed in an MHC class I-deficient cell line to demonstrate directly the cloned molecule's capacity to bind and present peptide Ag to CTL. These studies illustrate that AIDS virus-specific CTL can be characterized in detail in the rhesus monkey and lay the foundation for exploring novel approaches to AIDS virus vaccination in this species.

Amino Acid Sequence↗
PubMed1991

Immune response to HIV-1 gag antigens induced by recombinant adenovirus vectors in mice and rhesus macaque monkeys.

Recombinant adenovirus vectors expressing the entire gag (p55) or CA (p24) region of human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the appropriate HIV DNA sequences into the E3 region of human adenovirus type 5 (Ad5) with and without an exogenous SV40 early promoter. The infectious recombinant adenoviruses Adgag1, AdSVgag1, and AdSVCA1 were shown to express the appropriate HIV-1 antigens in human cells in vitro, as measured by immunoprecipitation and p24 antigen capture assays. Using the p24 antigen capture assay, HIV antigen expressed by AdSVCA1 was detected earlier in infection and in greater amounts than that produced by either Adgag1 or AdSVgag1. In studies concerning the immunogenicity of these vectors, Balb/c (H-2d) mice given a single intraperitoneal injection of 10(7) or 10(8) plaque-forming units of purified vector developed serum antibodies to p24, detected by Western blotting, by 2 weeks postinjection. In the preliminary test of the immunogenicity of the recombinant adenovirus vectors in primates, two of four rhesus macaque monkeys generated antibodies to HIV-1 p24 following two injections of AdSVCA1. As expected, monkeys injected with control adenovirus failed to show any anti-HIV response, and none of the monkeys showed any adverse reactions following infection with either recombinant or control adenoviruses. These results suggest that adenovirus vectors have considerable potential in the study of possible immune therapies for HIV infection.

Adenoviruses, Human↗
PubMed1991

Detection of HTLV-1 gag related sequences in leucocyte DNA from patients with polyendocrinopathies (Basedow-Graves' disease and insulin-dependent diabetes).

Relationships with retroviruses have recently been found in different human pathologies as autoimmune diseases which would be associated with the presence and eventually the expression of retroviral sequences. Detection of the presence of HTLV-1 and HIV-1 homologous sequences and their expression was realised on lymphocytes of 14 patients with polyendrocrinopathies (Basedow-Graves' disease and insulin-dependent diabetes) and four relatives of one index case. No antibodies to HTLV-1 and HIV-1 could be detected by Western blot and Elisa tests. HTLV-1 related sequences were revealed by Southern blot (SB) in 5 out of 18 subjects' DNA. Analyses of all DNA were performed by polymerase chain reaction (PCR). Seven DNA, including the 5 previously positive in SB, and two relatives (father and grandfather), negative in SB, contained HTLV-1-gag related sequences, but neither pol nor pX regions. Concerning HIV-1, all 18 DNA examined were negative by both methods. DNA of ten clinically healthy donors were found to be negative with the same tests.

Aged↗
PubMed1991

[Study by NMR and circular dichroism of synthetic "zinc finger" peptide of viral Gag protein from HIV-2].

We report the synthesis in solid phase of an 18-amino acid peptide that contains the cysteine-rich region of the structural Gag protein of HIV-2. The characterization of this fragment and of its interaction with Zn2+ has been made by one- (1 D) and two-dimensional (2D) NMR techniques and by circular dichroism. Our results suggest that in aqueous solution the complexation produces a significant perturbation in the conformation of this peptide.

Circular Dichroism↗
PubMed1989

Human monoclonal antibodies to HIV-1: cross-reactions with gag and env products.

Human monoclonal antibodies were raised by in vitro immunization of normal human spleen cells with denatured HIV-1 and subsequent fusion to an Epstein-Barr virus (EBV) transformed cell line. Three monoclonals reacted with both gag-encoded p24 core protein and env-encoded gp41 transmembrane glycoprotein. The cross-reaction was confirmed by reactivity with p24- and gp41-derived recombinant peptides. The peptides share two amino acid sequences which may be the basis for the cross-reaction. Attempted epitope mapping using synthetic peptides was unsuccessful due to non-specific reactivity with the short linear peptides.

Antibodies, Monoclonal↗
PubMed1989

V-sis gene (SSV) is expressed transiently and independently of v-gag (SSAV) after infection of fibroblasts with SSV/SSAV.

To investigate the mechanism of cell transformation by the retroviral v-sis gene, we examined the mode of its mRNA expression after infection of primate fibroblasts with Simian Sarcoma Virus (SSV/SSAV). Surprisingly transient expression of the 5.3 kb transcript of v-sis was detected between day two and four after infection. Addition of cycloheximide did not reverse the down-regulation of v-sis expression. Suramin, which uncouples the PDGF receptor complex, had no effect on the pattern of v-sis expression. A marginal but non-transient expression of c-myc and c-fos mRNA upon v-sis expression was detected. Studies on nuclear run-off and m-RNA stability suggest that the half-life time of v-sis mRNA is about 8 h or longer and that its expression is controlled rather by transcriptional than by post-transcriptional mechanisms. The up and down regulation of v-sis expression is independent of the expression of the helper virus (SSAV) gag-genes. This indicates that v-sis oncogene (SSV) and structural genes of the helper virus (SSAV) are obviously under separate expression control.

Animals↗
PubMed1989

Antibodies to structural and nonstructural gag-coded proteins of type-D retroviruses in humans with lymphadenopathy and AIDS.

The sera of patients with lymphadenopathies (children, suffering from primary immune deficiencies) and AIDS were analysed for the presence of antibodies against D-type retroviruses by means of radioimmunoprecipitation (RIP) and Western blotting. It was established that these sera contain the antibodies derived against p27, the major structural protein of type-D retroviruses, in a considerable percentage (12/80 for lymphadenopathy (LAP) patients and 9/12 for AIDS patients). Some serum samples reacted in RIP with Pr78gag, the precursor of gag-coded proteins of these viruses. The results obtained for AIDS patients are in contrast with those reported by other research groups. Nevertheless, it seems highly probable that D-type retroviruses are truly associated with human immunodeficiencies, as has been shown for simian ones.

AIDS-Related Complex↗
PubMed1985

Cell surface antigens of chemically induced fibrosarcomas: detection by a monoclonal antibody of a tumor-restricted Mr 12,000 protein gag antigen encoded by a dual-tropic murine leukemia virus.

Fusion products of spleen cells of W/FuDp rats immunized with a methylcholanthrene-induced BALB/c sarcoma, CA-2, and mouse myeloma cells were screened in an attempt to identify a monoclonal antibody defining the individually distinct tumor-specific transplantation antigen of CA-2. A hybridoma, MP/69/04, was isolated which produces an IgG2a monoclonal antibody that recognized a tumor-restricted antigen of CA-2. In direct binding assay, MP/69/04 reacted only with 2 of 15 methylcholanthrene induced BALB/c sarcomas tested. Thymus, spleen, lymph nodes, bone marrow, brain, adult lung fibroblasts, newborn muscle fibroblasts and 3T3 cells were negative. Absorption tests revealed, however, expression of the MP/69/04 determinant on 8 of the 12 murine leukemia virus (MuLV) producer BALB/c sarcoma tested. The antigen was not detected on any of the three non-producer sarcomas tested nor on a wide range of normal tissues and cell lines. An N-dualtropic MuLV was isolated from CA-2, and cell lines susceptible to infection by this virus were shown to express the MP/69/04 epitope. By Western blotting, the MP/69/04 epitope was identified as being expressed on the MuLV structural protein with a molecular weight of 12,000, present in CA-2 cells and in the purified CA-2 MuLV. These results indicate the MP/69/04 antigen is not a unique tumor-specific transplantation antigen but is a gag product of a recombinant retrovirus which is expressed on the cell surface of many MuLV + methylcholanthrene-induced BALB/c fibrosarcomas.

Animals↗
PubMed1986

A monoclonal antibody against LAV gag precursor: use for viral protein analysis and antigenic expression in infected cells.

A murine monoclonal antibody (MAb), named C.V.K., was produced after immunization with highly purified and sonicated lymphadenopathy-associated virus (LAV). No monoclonal antibody was observed with intact virus used as immunogen. C.V.K. MAb recognizes an epitope present on the precursor gag protein of 55 kilodaltons. Western blot analysis and pulse-chase experiments support the interpretation that after p55 cleavage into p25, p18, and p13, only p18 expresses this epitope. C.V.K. MAb selectively stained only LAV-infected lymphocytes. This intracytoplasmic staining appears 3 days after the infection and is correlated with reverse transcriptase activity. Neither membrane immunofluorescence of infected lymphocytes nor neutralizing activity was observed with C.V.K. MAb. These facts suggest that p55 and p18 are not expressed at the cell membrane or on the viral envelope. C.V.K. MAb should prove useful not only in the purification of core proteins but also for detecting infected cells producing the virus in suspension or on histologic sections.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Genetic structure of the endogenous proviruses and expression of the gag gene in Brown Leghorn chickens.

Seven loci of endogenous proviruses were detected in the genome of Brown Leghorn chickens. Sets of endogenous proviruses in DNA of the chicken embryos examined were identified by blot hybridization with 32P-labelled DNA of RSV and EcoRI restriction endonuclease digestion. Comparison of the results showed that only one locus (A) of endogeneous provirus was associated with a gs+ phenotype as determined by the immunoperoxidase reaction and antibodies against gag gene products of RSV. Restriction endonuclease analysis with HindIII, BamHI and SacI revealed that proviruses A and F in Brown Leghorn chickens correspond to loci ev-3 and ev-6, respectively, in White Leghorn chickens. Other loci (B, C, D, E, and X) were designated ev-22, ev-23, ev-24, ev-25, ev-26, respectively. None of these loci expressed infectious virions. The structure of most of the endogenous proviruses examined is considerably different from the genome of the endogenous chicken virus RAV-O. The difference in structure may be one possible cause of the absence of endogenous provirus expression.

Animals↗
PubMed1986

[2 nonstructural glycosylated polyproteins coded by the gag gene of retrovirus type D].

Two nonstructural glycosylated gag-encoded polyproteins were identified in D type retrovirus-infected cells of human transplantable laryngeal carcinoma by a "pulse-chase" modification of the immunoprecipitation method. The molecular weights of these polyproteins were 78 K (gPr 78gag) and 90 K (gPr 90gag), respectively. Glucosamine and galactose constituted their carbohydrate component. Initially, gPr 90gag is synthesized, to be followed by gPr 78gag, perhaps due to the split of gPr 90gag. The functional role of these glycoproteins remains to be established. It is suggested that the said and similar glycosylated polyproteins of other retroviruses play a role in stimulating proliferation of retrovirus-infected cells.

Chemical Precipitation↗
PubMed1984

Analysis of gag-myc proteins of partially transformation-defective mutants of avian myelocytomatosis virus strain MC29 by in vitro cleavage with protease p15: indication for the presence of specific cleavage site p15 in the myc domain of fusion protein.

Gag-related proteins were immunoprecipitated from [35S]methionine-labelled cells of the PR-2 line which contained p110 protein as well as from quail cells transformed by deletion mutants 10A, 10C, and 10H of MC29 virus. Immunoprecipitates were incubated with oncoviral protease p15 and cleavage products were analyzed in SDS-PAGE. The major 56K fragment (F56) of p110 was further analyzed by tryptic peptide mapping. The results showed that except for the myc domain of p110, a portion of p27 is also present in F56. Cleavage of 100K, 95K, and 90K proteins coded by three MC29 deletion mutants resulted in major fragments 66K, 60K, and 56K, respectively. The existence of further cleavage fragments and presence of the p15 specific cleavage site in the myc domain of MC29 specific proteins is discussed.

Animals↗
PubMed1982

Polypeptide composition and gag gene-coded products of type-D oncovirus from HEp-2 cells.

The protein composition of type-D oncovirus HEp-2, isolated from cell-free medium of continuous human HEp-2 cell line, has been investigated using electrophoresis on gradient polyacrylamide gels with sodium dodecyl sulfate (SDS). Labeling with 14C-amino acids revealed five viral polypeptides with molecular weights of 70 000 (gp70), 27 000 (p27), 19 000 (p19), 15 000 (p15), 12 000-10 000 (p12-10). The 70 000 dalton protein was shown to be the only glycoprotein by incorporation of radioactive glucosamine. A polypeptide with molecular weight of 78 000 has been specifically precipitated from pulse-labeled type-D oncovirus producing HEp-2 cells with goat anti Mason-Pfizer p27 serum. This protein was shown to be gag gene-coded polyprotein precursor (Pr78gag) of the major virus polypeptide p27. Pulse-labeled HEp-2 and Mason-Pfizer infected Tu 197 cells were rinsed, lysed, clarified and precipitated with goat anti Mason-Pfizer p27 serum. In both cases Pr78gag was detected.

Gene Products, gag↗
PubMed1995

Analysis of the role of the pseudoknot component in the SRV-1 gag-pro ribosomal frameshift signal: loop lengths and stability of the stem regions.

The simian retrovirus-1 (SRV-1) gag-pro frameshift signal was identified in previous work, and the overall structure of the pseudoknot involved was confirmed (ten Dam E, Brierley I, Inglis S, Pleij C, 1994, Nucleic Acids Res 22:2304-2310). Here we report on the importance of specific elements within the pseudoknot. Some mutations in stem S1 that maintain base pairing have reduced frameshift efficiencies. This indicates that base pairing in itself is not sufficient. In contrast, frameshifting correlates qualitatively with the calculated stability of mutations in S2. The stems thus play different roles in the frameshift event. The nature of the base in L1 has little influence on frameshift efficiency. It is however required to bridge S2; deleting it lowers frameshifting from 23 to 9%. In L2, frameshift efficiency was not affected in a mutant that changed 10 to 12 bases. This makes it unlikely that the primary sequence of L2 plays a role in -1 frameshifting, in contrast to readthrough in Moloney murine leukemia virus (Wills N, Gesteland R, Atkins J, 1994, EMBO J 13:4137-4144). Deletions of 2 and 3 bases gave more frameshifting than the wild type, probably reflecting the increased stability of the pseudoknot due to a shorter loop L2. Deleting even more bases reduces frameshifting compared to wild-type levels. At this point, stress will build up in L2, and this will reduce overall pseudoknot stability.

Animals↗