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PubMed1995

Construction of gag, pol, and env specific riboprobes for confirmation of HIV-1 specific polymerase chain reaction products.

Conserved regions of the gag, pol, and env genes of HIV-1 pBH10 DNA (gag nucleotides(nt)1508-1652, pol nt 2811-3118, env nt 7792-7934; Ratner et al., 1985) were amplified by the polymerase chain reaction (PCR) using oligonucleotides complementary to the termini of these regions as primers. Primer areas of the amplified DNA were then removed by digestion with restriction endonucleases, and the internal fragments purified and cloned in both orientations into the 'riboprobe' transcription vector pGEM-5Z. Riboprobes made from these plasmids did detect the specific sequences of pBH10 DNA and of HIV-1 DNA amplified by PCR from clinical material. The riboprobes will be useful to confirm the specificity of PCR-amplified fragments of lymphocyte DNA obtained from infants of HIV-infected mothers and from high risk, but seronegative contacts of HIV-1 infected individuals.

Cloning, Molecular↗
PubMed1994

Construction of gag, pol, and env specific riboprobes for confirmation of HIV-1 specific polymerase chain reaction products.

Conserved regions of the gag, pol, and env genes of HIV-1 pBH10 DNA (gag nucleotides(nt)1520-1665, pol nt 2326-2663, env nt 7192-7333; Ratner et al., 1985) were amplified by the polymerase chain reaction (PCR) using oligonucleotides complementary to the termini of these regions as primers. Primer areas of the amplified DNA were then removed by digestion with restriction endonucleases, and the internal fragments purified and cloned in both orientations into the 'riboprobe' transcription vector pGEM-5Z. Riboprobes made from these plasmids did detect the specific sequences of pBH10 DNA and of HIV-1 DNA amplified by PCR from clinical material. The riboprobes will be useful to confirm the specificity of PCR-amplified fragments of lymphocyte DNA obtained from infants of HIV-infected mothers and from high risk, but seronegative contacts of HIV-1-infected individuals.

DNA, Viral↗
PubMed1992

Myristoylation-dependent membrane targeting and release of the HTLV-I Gag precursor, Pr53gag, in yeast.

The unprocessed HTLV-I Gag precursor, Pr53gag, was synthesized in yeast, Saccharomyces cerevisiae. The synthesized Pr53gag was myristoylated, associated with the cellular membrane, and released into the culture medium. The released Pr53gag was pelleted by centrifugation at 100,000 x g for 2 h. Conversion of Gly2 to Ala allowed synthesis of a non-myristoylated soluble Pr53gag which was not released into the culture medium. These results suggest that the release of the HTLV-I Gag precursors Pr53gag, occurs in yeast in a myristoylation-dependent manner.

Amino Acid Sequence↗
PubMed1990

Detection and quantitation of a type D retrovirus gag protein in ovine pulmonary carcinoma (sheep pulmonary adenomatosis) by means of a competition radioimmunoassay.

A heterologous competition radioimmunoassay (RIA) which consisted of 125I-labeled langur retrovirus major gag protein and goat anti-squirrel monkey retrovirus serum was used to detect a type D retrovirus-associated antigen in tumor cell homogenates, lung fluid, and cell culture supernatant fluids of naturally occurring and experimentally-induced ovine pulmonary carcinoma (OPC, sheep pulmonary adenomatosis). In this assay, there was no cross reactivity between structural proteins of the type D retrovirus and an ovine lentivirus, which frequently co-infects OPC-affected sheep. The sensitivity of the assay was similar to an immunoblotting assay using antiserum to Mason-Pfizer monkey virus major gag protein which had been used previously to detect the OPC retrovirus antigen in tumor homogenates and lung fluids of OPC-affected sheep. All unconcentrated samples of lung fluid collected from five sheep with naturally occurring OPC or six sheep with experimentally induced OPC competed in the competition RIA. The competition RIA titers of the type D retrovirus antigen in lung fluids of lambs with induced OPC were relatively higher than the titers of this antigen in the naturally occurring OPC cases. The competition RIA detected the retrovirus antigen associated with OPC in the culture fluids of four out of five primary lung cultures from OPC sheep tested between 1 and 56 days after culture initiation. Because this RIA is appropriate for the quantitation of OPC-associated antigen, it will provide a means for determination of the target cell type for OPC virus replication in vitro.

Animals↗
PubMed2004

HLA-A and -B allele expression and ability to develop anti-Gag cross-clade responses in subtype C HIV-1-infected Ethiopians.

A cohort of 35 human immunodeficiency virus type 1 (HIV-1) subtype C-infected Ethiopians was studied to define the HLA phenotype in all 35 subjects and highly conserved Gag protein regions involved in cross-clade cell-mediated immunity. Full-length Gag virus sequences were determined in 15 individuals. CD8 cell-mediated immune responses were detected by interferon-gamma ELISpot assay. HLA-A*03, -B*49, and -B*57 allelic frequencies were relatively higher than in other African populations. Anti-p17 (aa 1-60) CD8+ were detectable in the highest number of individuals. Anti-p17 (aa 1-60 and 51-110) cross-clade responses against subtype B and C were detected in 50% of the tested subjects. The p24 KF11 (aa 162-172) epitope was found to be immunodominant among the HLA-B*5703--positive individuals. These data represent the first report of correlating HLA phenotype and HIV-specific cell-mediated immune responses among infected Ethiopians and may be useful in designing cytotoxic T lymphocyte-inducing vaccines for this part of Africa.

AIDS Vaccines↗
PubMed2005

Expression and characterization of Gag protein of HIV-1(CN) in Pichia pastoris.

To express the core protein of HIV-1 of Chinese prevalent strain (HIV-1(CN)) in Pichia pastoris, the full-length gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by SalI was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA (1.3kb) was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55kDa, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13% of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows: BMMY medium, 80-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85%. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.

Animals↗
PubMed2005

A genotypic assay for the amplification and sequencing of gag and protease from diverse human immunodeficiency virus type 1 group M subtypes.

In human immunodeficiency virus type 1 (HIV-1), an interaction exists between the in vivo evolution of Gag protein and protease to escape from antiretroviral drug selective pressure. Therefore, it was decided to develop a genotypic assay for the amplification and sequencing of HIV-1 gag and protease. As the HIV-1 pandemic is characterised by a large genetic diversity, the assay developed was evaluated on a panel of 28 genetically divergent samples belonging to the following subtypes A1, B, C, D, F1, F2, G, H, J, CRF01-AE, CRF02-AG and CRF13-cpx. The assay displayed a detection limit ranging between 500 RNA copies/ml and 5000 RNA copies/ml plasma. Full-length sequences could be obtained for 25 samples. The population sequences of the three other samples lacked a part of the sequence because of heterogeneous signal, probably due to the presence of quasi-species with insertions/deletions of a different length.

DNA Primers↗
PubMed2006

Both the 5' and 3' LTRs of FIV contain minor RNA encapsidation determinants compared to the two core packaging determinants within the 5' untranslated region and gag.

This study was undertaken to address the role of feline immunodeficiency virus (FIV) long terminal repeats (LTR) as potential packaging determinants. A number of studies in the recent past have clearly demonstrated that the core packaging determinants of FIV reside within at least two distinct regions at the 5' end of the viral genome, from R in the 5' LTR to approximately 150 bp within the 5' untranslated region (5' UTR) and within the first 100 bp of gag; however, there have been conflicting observations as to the role of the LTR regions in packaging and whether they contain the principal packaging determinants of FIV. Using a semi-quantitative RT-PCR approach on heterologous non-viral vector RNAs in an in vivo packaging assay, this study demonstrates that the principal packaging determinants of FIV reside within the first 150 bp of 5' UTR and 100 bp of gag (the two core regions) and not the viral 5' LTR. Furthermore, it shows that in addition to the 5' LTR, the 3' LTR also contains packaging determinants, but of a less significant nature compared to the core packaging determinants. This study defines the relative contribution of the various regions implicated in FIV genomic RNA packaging, and reveals that like other primate lentiviruses, the packaging determinants of FIV are multipartite and spread out, an observation that has implications for safer and more streamlined design of FIV-based gene transfer vectors.

3' Untranslated Regions↗
PubMed2006

Construction of gag-chimeric viruses between HIV-1 and SIVmac that are capable of productive multi-cycle infection.

Forty-nine recombinant viral clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus from the rhesus monkey (SIVmac), which carry chimeric gag (capsid/p2 region) genes in the background of the HIV-1 genome, were constructed to establish an HIV-1/monkey infection model system for human AIDS. Upon transfection, all the recombinants generated progeny virions at a level comparable to the parental HIV-1 clone and no major abnormalities were found in the virions, as examined by Western blot analysis. In infection experiments, 18 recombinants grew in human lymphocytic cells and six of these clones propagated as well as the parental virus, as monitored by virion associated-reverse transcriptase production. By contrast, none of the recombinants grew at a detectable level in monkey lymphocytic cells. The defective replication site(s) in human cells for non-infectious recombinants was mapped to the step before and/or during reverse transcription. Our results described here showed that HIV-1 type chimeric viruses between HIV-1 and SIVmac, which are capable of spreading productive infection, are readily constructed throughout the capsid/p2 region. In addition, it is suggested that there may be a viral determinant(s), other than Gag, responsible for the species-specific tropism of HIV-1 and which is associated with viral DNA synthesis.

Animals↗
PubMed2006

Novel codon-optimized GM-CSF gene as an adjuvant to enhance the immunity of a DNA vaccine against HIV-1 Gag.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine. Here we generated a novel codon-optimized murine GM-CSF gene as an adjuvant. The codon-optimized GM-CSF gene significantly increased protein expression levels in all cells tested. Although injection of the wild-type GM-CSF plasmids adjuvanted HIV-1 Gag DNA vaccine induced detectable immune responses, co-administration of plasmids encoding the codon-optimized GM-CSF sequence with the DNA vaccine resulted in a strong antibody and CTL responses and a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag. This novel codon-optimized GM-CSF gene offers a practical molecular strategy for potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

AIDS Vaccines↗
PubMed2006

Comprehensive epitope analysis of cross-clade Gag-specific T-cell responses in individuals with early HIV-1 infection in the US epidemic.

To elucidate the mechanisms underlying cross-clade T-cell reactivity, we evaluated responses to Gag peptides based on clades A, B, C, and M-group sequences at the epitope level by IFN-gamma ELISpot assay in 25 subjects following primary clade B infection. T-cell reactivity to CON (consensus), COT (center of tree), and ANC (most recent common ancestor) B peptides was similar and a high level of cross-reactivity was noted to clade A, C, and M-group peptides. T-cell responses to 15 of the 16 epitopes reacted with at least 1 of the 2 heterologous peptides (A or C or both) and 7 epitopes were invariant across all 3 clades. The remaining 9 epitopes were associated with a total of 11 variant sequences, and with the exception of 1, all substitutions were outside the HLA anchor positions. We conclude that Gag-specific cross-clade T-cell responses producing IFN-gamma can be detected in primary HIV-1 infection. Cross-reactivity is attributable to the recognized epitopes being either invariant across clades or differing by single amino acid substitutions outside the HLA anchor sites. Semi-conservative and non-conservative substitutions that presumably involve the T-cell receptor contact sites have significant effects on T-cell recognition. Finally, further studies are needed to determine if the detection of cross-clade IFN-gamma T-cell responses indeed translates to cross-reactive antiviral activity.

Acquired Immunodeficiency Syndrome↗
PubMed2005

The T12I mutation within the SP1 region of Gag restricts packaging of spliced viral RNA into human immunodeficiency virus type 1 with mutated RNA packaging signals and mutated nucleocapsid sequence.

Specific packaging of human immunodeficiency virus type 1 (HIV-1) RNA is attributable to the high affinity of nucleocapsid (NC) sequence of Gag for the cis-acting RNA packaging signals located within the 5' un-translated region (5' UTR). Interestingly, we have previously reported that the T12I mutation (named MP2) within SP1 of Gag prevented incorporation of spliced viral RNA into mutated viruses that lacked the stem-loop 1 (SL1) RNA element (also named dimerization initiation site, DIS), suggesting a role for the SP1 sequence in viral RNA packaging. In this study, we have further tested this activity of MP2 in the context of a variety of mutations that affect viral RNA incorporation. The results showed that MP2 was able to effectively restrict packaging of spliced viral RNA into viruses containing either NC mutations R10A and K11A or mutated 5' UTR sequence, such as DeltaGU3 that lacked the 112-GUCUGUUGUGUG-123 sequence of U5, D1 that was deleted of a 27 nt fragment immediately downstream of the primer binding site (PBS), Delta(306-325) that had the SL3 RNA element removed and MD2 that was missing the 328-GGAG-331 sequence. As a result, MP2 contributed increased infectivity to the related viruses. Therefore, the MP2 mutation demonstrates a distinct role in HIV-1 RNA packaging that is neither pertained to the specific viral RNA packaging signal nor to the NC sequence.

Animals↗
PubMed2003

Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin.

The host range of swinepox virus (SPV) is restricted to swine, although SPV has been shown to infect mammalian, non-swine cells, without recovery of infectious virus. SPV is a reasonable candidate for development as a non-productively replicating viral vector for use in non-swine, mammalian species, such as the cat. A novel SPV gene deletion (SPV 043) was created and found to be non-attenuating. This deletion was utilized to generate a stable recombinant virus expressing the Gag-Pro and Env proteins of feline leukemia virus (FeLV). Expression and replication of this vector was studied in embryonic swine kidney cells (ESK-4), and two feline cell lines, Crandell feline kidney cells (CRFK) and feline skin fibroblasts (FSF). Our results showed that feline cells were susceptible to infection by SPV and supported expression of foreign genes driven by synthetic poxvirus promoters, however, SPV viral DNA was not replicated in feline cells and infectious virus was not recovered. In addition, FeLV Gag virus-like particles were produced from both ESK-4 and CRFK cells and foreign antigens were incorporated into infectious SPV intracellular mature virions (IMV). These results suggest that SPV may have potential as a safe vaccine delivery vector for cats.

Animals↗
PubMed2003

Role of HIV-1 Gag domains in viral assembly.

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.

Amino Acid Sequence↗
PubMed1988

HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins.

The mature proteins of retroviruses originate as a result of proteolytic cleavages of polyprotein precursors. Retroviruses encode proteases responsible for several of these processing events, making them potential antiviral drug targets. A 99-amino acid HIV-1 protease, produced by chemical synthesis or by expression in bacteria, is shown here to hydrolyze peptides corresponding to all of the known cleavage sites in the HIV-1 gag and pol polyproteins. It does not hydrolyze peptides corresponding to an env cleavage site or a distantly related retroviral gag cleavage site.

Amino Acid Sequence↗
PubMed1997

Cleavage of Gag precursor is required for early replication phase of HIV-1.

A mutant of human immunodeficiency virus type 1 (HIV-1), which is deficient for Gag precursor cleavage and noninfectious, was characterized with respect to its defective step in the viral replication phase. Upon transfection, the mutant produced a normal level of progeny virions as monitored by electron microscopy and RNA hybridization. Single-round replication assay demonstrated, in contrast, that the mutant was defective at the early phase of the replication cycle. Furthermore, no viral DNA was detected in the cells infected with the mutant. Taken together, it is concluded that maturation of Gag precursor protein of HIV-1 is required for an early event(s) before or during a coupled process of uncoating/reverse transcription.

Blotting, Southern↗
PubMed1999

Binding of oligopyrimidines to the RNA hairpin responsible for the ribosome gag-pol frameshift in HIV-1.

The 12 bp stem of the RNA hairpin responsible for the gag-pol frameshifting of the ribosomes during translation of the polycistronic HIV-1 mRNA has a pyrimidine-rich 5' strand and, consequently, a purine-rich 3' strand. Electrophoretic mobility shift assays have shown that DNA oligopyrimidines, 12 and 20 nucleotides long (but not oligopurines or G,T-containing oligomers), designed to form triplexes actually bind to the double-stranded RNA target. RNase V1 footprinting studies have confirmed the interaction between the hairpin stem and the RNA and 2'-O-methyl oligoribonucleotide analogues of the 12-mer oligodeoxypyrimidine as well as 5 propynyl,cytosine, containing the 12-mer oligodeoxypyrimidine, bind more strongly to the RNA target than the unmodified parent DNA oligomer. The complexes formed by the RNA hairpin and either the 12-mer oligodeoxypyrimidine or the 20-mer oligopyrimidine are stable at a neutral pH and in the absence of Mg2+ but blocked neither the reverse transcription nor cell-free translation of a RNA template in which the gag-pol frameshifting hairpin was inserted at the 5' end of the luciferase open reading frame.

Biological Assay↗
PubMed2003

Intragenic HIV-1 env sequences that enhance gag expression.

Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function.

Base Sequence↗