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At least 73 records · Page 4Linked to original sources
PubMed1998

Loss of viral fitness associated with multiple Gag and Gag-Pol processing defects in human immunodeficiency virus type 1 variants selected for resistance to protease inhibitors in vivo.

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.

Cell Line, Transformed↗
PubMed2005

Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens.

We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/K(b) transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8+ T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolDeltaFsDeltaPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8+ T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolDeltaFsDeltaPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses.

Amino Acid Sequence↗
PubMed1988

Antibodies reactive with human immunodeficiency virus gag-coded antigens (gag reactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population.

Normal blood donors were examined for human immunodeficiency virus (HIV)-reactive antibodies with both virus- and Escherichia coli-expressed env- and gag-coded antigens. The frequency of samples from normal (low-risk) donors that were repeatedly reactive with an HIV enzyme-linked immunosorbent assay blood screening test (Du Pont Co.) was 0.6%. Two classes of HIV serological reactivity were identified: a minor env-reactive class (0.03 to 0.06% of donors) and the predominant env-nonreactive gag-reactive class (gag reactive only [GRO]) (0.4 to 0.5% of donors). Assignment of env reactivity was made by a synthetic (recombinant) env enzyme-linked immunosorbent assay and virus immunoblot. Most GRO sera reacted with p15/p17 bands on HIV immunoblot. Antibody specificity in GRO sera was confirmed by competition-binding studies with viral gag and E. coli-expressed p55gag. This study provides independent verification that gag-specific antibodies are present in many env-nonreactive sera. More serological and virological studies of individuals with this antibody pattern should be pursued to determine the origin of these gag-reactive antibodies.

Antibodies, Viral↗
PubMed2002

Tsg101, an inactive homologue of ubiquitin ligase e2, interacts specifically with human immunodeficiency virus type 2 gag polyprotein and results in increased levels of ubiquitinated gag.

The final stages of budding and release of a retroviral particle from the cell require the late (L) domain of Gag. Recently, ubiquitin and ubiquitin ligases have been implicated in the late stages of retroviral budding. In a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that interact with human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified Tsg101, an inactive homologue of ubiquitin ligase E2. Tsg101 and HIV-2 Gag interact specifically in vitro and in vivo. The interaction requires the L domain PTAPP motif in the p6 domain of HIV-2 Gag and the N-terminal Ubc-conjugation homology domain of Tsg101. Tsg101 is incorporated into HIV-2 virions. Expression of the N-terminal Ubc-conjugation homology domain of Tsg101 inhibits the release of HIV-2 virus particles. Overexpression of Tsg101 results in an increase in the level of ubiquitination of HIV-2 Gag. Our results provide evidence for recruitment of the ubiquitination machinery of the cell during late stages of the viral life cycle, mediated by the viral Gag protein.

Blotting, Western↗
PubMedDate not supplied

Acupuncture/acupressure to treat gagging dental patients: a clinical study of anti-gagging effects.

Noninvasive methods advocated by some clinicians are not very successful in dealing with patient gagging, while sedation approaches run the risk of the patient aspirating foreign bodies. The P-6 Neikuan acupuncture point, located on the wrist, has been used in the Far East for thousands of years for its anti-nausea and anti-anxiety properties. In the West, it is the acupuncture point most studied. Only recently has interest been shown in the P-6 point for its possible anti-gagging effect. The anti-gagging effect of P-6 stimulation is documented in this article. The P-6 point has remarkable anti-gagging effects if stimulation is applied correctly. Clinicians may apply thumb pressure at the P-6 point to achieve some effect, although this is not as effective as acupuncture. Nevertheless, a substantial percentage of gagging patients would be able to go through dental procedures without gagging when the P-6 point is stimulated.

Acupressure↗
PubMed1997

CM-gag, a transposable-like element reiterated in the genome of Culex pipiens mosquitoes, contains only a gag gene.

CM-gag elements constitute an homogeneous family of sequences that are reiterated in the genome of Culex pipiens strains from different continents. Apparently complete 1.75 kb CM-gag copies are flanked by target-site duplications and have a polyadenylation signal near their 3' end. They potentially contain a unique gene encoding a putative protein that displays homologies with nucleic acid binding proteins and the gag polypeptide of retroviruses and retrotransposons, but that does not encode a reverse transcriptase. CM-gag elements are similar in their genetic organization to the telomeric transposable sequences Het-A from Drosophila melanogaster, but Southern-hybridization patterns indicate that the former are more probably dispersed in various areas of the mosquito genome. The homogeneity of CM-gag copies that are distributed worldwide suggests that they have most probably been amplified recently. Furthermore, selective constraints against amino acid changes have been acting on these sequences, suggesting that they need to encode the gag-like protein to be incorporated into the chromosomes.

Amino Acid Sequence↗
PubMed1999

A human cell line constitutively expressing HIV-1 Gag and Gag-Pol gene products.

A human cell line constitutively expressing the HIV-1 gag and pol genes products was established. The cell line was established by stably transfecting 293 cells with a plasmid construct that expresses the HIV Gag and Pol and can confer the transfectants resistant to mycophenolic acid. Particles generated from transient expression of the plasmid construct were noninfectious when pseudotyped with HIV envelope or with amphotropic murine leukemia virus envelope proteins. However, virus-like Gag particles produced by the stable cell line were appropriately processed, exhibited a wild-type retrovirus particle density, and possessed significant reverse-transcriptase (RT) activities. Continuous passage of the cell line either in the presence or absence of mycophenolic acid had no major effects on the Gag processing efficiency, particle assembly, or RT activity release. It was also demonstrated that the proteolytic processing of the virus-like particles released from the cell line was inhibited by an HIV protease inhibitor, saquinavir. The establishment of a stable cell line producing noninfectious but proteolytically processed HIV Gag particles offers a safe, convenient tool for biochemical and immunological analysis of virus-like particle assembly and is very useful for the development of anti-HIV protease drugs.

Blotting, Western↗
PubMed1998

Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells.

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.

Adult↗
PubMed2005

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses.

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.

AIDS Vaccines↗
PubMed1994

Site-directed mutagenesis of the P2 region of the Rous sarcoma virus gag gene: effects on Gag polyprotein processing.

The Pr76Gag and Pr180Gag-Pol polyprotein precursors of Rous sarcoma virus contain a 22-amino-acid spacer peptide, called p2, located between the amino acid sequences of the mature Gag proteins MA and p10. This spacer peptide is present in stoichiometric amounts in the virion, albeit cleaved into two parts, but its function is unknown. The primary sequence of this peptide includes a region that is highly conserved among retroviruses, consisting of four prolines followed by tyrosine. We have investigated the role of p2, particularly the polyproline motif, in the virus life cycle by site-directed mutagenesis. Mutations in this region result in the intracellular accumulation of a truncated Gag precursor, due either to a block in the intracellular processing of the precursor or to the premature activation of the viral protease. Since in cells infected by Rous Sarcoma Virus there is no significant intracellular processing of the Gag polyprotein precursor, our data suggest that the p2 domain plays a role in controlling the activation of the protease. These mutations also result in a reduction in virus particle release, probably as a direct consequence of the aberrant precursor processing since a construct in with both p2 and the protease active site were mutated did not exhibit aberrant processing of the Gag polyproteins and formed particles with an efficiency similar to that of the wild type. This indicates that it is the viral protease that is responsible for the aberrant processing and suggests that the p2 region is not required for assembly. Although the virus genomic RNA packaged into virions produced by the p2 mutants is more susceptible to degradation, it appears that the p2 domain does not have a direct role in RNA packaging and protection.

Amino Acid Sequence↗
PubMed1990

Effect of two novel inhibitors of the human immunodeficiency virus protease on the maturation of the HIV gag and gag-pol polyproteins.

The ability of two novel synthetic compounds to inhibit the HIV protease-mediated processing of HIV-1 precursor polyproteins was investigated in an in vitro gag-protease mixed lysate assay system and in an assay using recombinant baculoviruses engineered to express the HIV-1 gag and pol genes in cultured insect cells. With the in vitro mixed lysate assay we have shown that both compounds at 1 microM can completely inhibit the HIV-1 and HIV-2 protease-mediated release of p24 from the HIV-1 gag precursor at pH 5.5 and pH 7.0. In the intracellular baculovirus system these compounds were shown to inhibit the protease-mediated maturation of gag and also the excision of the protease moiety from its precursor.

Animals↗
PubMed1992

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo.

Animals↗
PubMed1995

The Gag-Pol encoded proteinase of an avian retrovirus expressed in E. coli can produce a novel proteinase (PR + IleGly) that is two amino acids larger at its carboxy-terminal region than the major Gag proteinase (PR).

Gag-Pol frameshift translational products of avian retroviruses (e.g., myeloblastosis associated virus, MAV) contain a putative proteinase species of 131 amino acids that maps between the NC/PR and the PR/RT processing sites. Expression in Escherichia coli of an in-frame PR precursor that contains the natural NC/PR processing site and is translationally terminated at the PR/RT site leads to formation of a Gag-Pol proteinase of the expected molecular size (131 amino acids) and a novel PR product of 126 amino acids. This product extends 2 amino acids downstream of the gag-encoded 124 amino acids, and its proteolytic cleavage is promoted by conditions favorable for enzyme catalysis, is blocked by a specific MAV proteinase inhibitor, and can be demonstrated also for corresponding peptide substrates. The new self-processing cleavage product is termed PR(+IleGly) and exhibits similar, but slower, catalytic parameters than those of the Gag PR.

Amino Acid Sequence↗
PubMed1986

Avian sarcoma virus gag-fps and gag-yes transforming proteins are not myristylated or palmitylated.

The transforming proteins of several avian sarcoma viruses were examined for evidence of covalently attached fatty acids. While the product of the viral src gene could be readily labeled biosynthetically with [3H]myristic acid, the gag-onc transforming proteins of Fujinami sarcoma virus, PRCII, PRCIIp, and Y73 avian sarcoma viruses were not readily labeled with either [3H]myristate or [3H]palmitate. Thus, avian gag-onc proteins appear to lack modifications shared by mammalian gag and gag-onc proteins, and the products of the oncogenes src, tck, and ras.

Animals↗
PubMed1990

Epitope mapping of the HIV-1 gag region by analysis of gag gene deletion fragments expressed in Escherichia coli defines eight antigenic determinants.

Immune response to HIV infection is generally characterized by appearance of antibodies to the gag protein p24 early in infection, and by apparent loss of p24 antibodies accompanied by increases in p24 antigen levels with disease progression. Precise definition of the immunodominant epitopes present in gag gene proteins has potential clinical significance. Seventeen anti-gag monoclonal antibodies (MAb) were used in enzyme-linked immunosorbent assays (ELISA) with antigens expressed by nine recombinant clones to define epitopes on HIV gag proteins which elicit an immune response. All of the MAbs tested, except two anti-p17, reacted with a clone which expresses the carboxyl terminal 13 amino acids of p17 and all of p24 and p15. All anti-p24 MAbs reacted with clones containing all of p24. MAbs reacted differentially with clones containing deleted regions depending on the antigenic portion expressed. Of thirteen potential identifiably different genomic regions which could be predicted from the genomic structure of the clones, eight different antigen epitopes were defined: two on p17, five on p24, and one on p15 (in the region corresponding to the carboxyl terminal protein p6). Six regions did not appear to react with any of the monoclonal antibodies available. Identification of the epitopes present in the cloned antigens should allow their use to evaluate sera from HIV-infected donors at different clinical stages of progression to AIDS.

Animals↗
PubMed2004

Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice.

Human immunodeficiency virus type 1 (HIV-1) subtype C is the predominant HIV in southern Africa, and is the target of a number of recent vaccine candidates. It has been proposed that a heterologous prime/boost vaccination strategy may result in stronger, broader and more prolonged immune responses. Since HIV-1 Gag Pr55 polyprotein can assemble into virus-like particles (VLPs) which have been shown to induce a strong cellular immune response in animals, we showed that a typical southern African subtype C Pr55 protein expressed in insect cells via recombinant baculovirus could form VLPs. We then used the baculovirus-produced VLPs as a boost to a subtype C HIV-1 gag DNA prime vaccination in mice. This study shows that a low dose of HIV-1 subtype C Gag VLPs can significantly boost the immune response to a single subtype C gag DNA inoculation in mice. These results suggest a possible vaccination regimen for humans.

AIDS Vaccines↗
PubMed1993

Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Absence of immunodominant anti-Gag p17 (SL9) responses among Gag CTL-positive, HIV-uninfected vaccine recipients expressing the HLA-A*0201 allele.

According to a number of previous reports, control of HIV replication in humans appears to be linked to the presence of anti-HIV-1 Gag-specific CD8 responses. During the chronic phase of HIV-1 infection, up to 75% of the HIV-infected individuals who express the histocompatibility leukocyte Ag (HLA)-A*0201 recognize the Gag p17 SLYNTVATL (aa residues 77-85) epitope (SL9). However, the role of the anti-SL9 CD8 CTL in controlling HIV-1 infection remains controversial. In this study we determined whether the pattern of SL9 immunodominance in uninfected, HLA-A*0201 HIV vaccine recipients is similar to that seen in chronically HIV-infected subjects. The presence of anti-SL9 responses was determined using a panel of highly sensitive cellular immunoassays, including peptide:MHC tetramer binding, IFN-gamma ELISPOT, and cytokine flow cytometry. Thirteen HLA-A*0201 vaccinees with documented anti-Gag CD8 CTL reactivities were tested, and none had a detectable anti-SL9 response. These findings strongly suggest that the pattern of SL9 epitope immunodominance previously reported among chronically infected, HLA-A*0201-positive patients is not recapitulated in noninfected recipients of Gag-containing canarypox-based candidate vaccines and may be influenced by the relative immunogenicity of these constructs.

AIDS Vaccines↗