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At least 1,081 records · Page 60Linked to original sources
PubMed1992

Bipartite signal for read-through suppression in murine leukemia virus mRNA: an eight-nucleotide purine-rich sequence immediately downstream of the gag termination codon followed by an RNA pseudoknot.

The pol gene of murine leukemia virus and other mammalian type C retroviruses is expressed by read-through suppression of an in-frame UAG codon which separates the gag and pol coding regions. In this study, we have analyzed the sequence requirements for read-through suppression by placing different portions of wild-type and mutant viral sequences from the gag-pol junction between reporter genes and testing transcripts of these constructs for suppression in reticulocyte lysates. We find that the read-through signal is contained within the first 57 nucleotides on the 3' side of the UAG codon. Our results indicate that the identities of six conserved bases in the eight-nucleotide, purine-rich sequence immediately downstream of the UAG codon are critical for suppression, as is the existence of a pseudoknot structure spanning the next 49 nucleotides. Thus, read-through suppression depends on a complex, bipartite signal in the mRNA.

Animals↗
PubMed1992

Human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo.

The human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion polyprotein is produced via ribosomal frameshifting. Previous studies in vitro and in Saccharomyces cerevisiae have argued against a significant role for RNA secondary structure 3' of the shift site, in contrast with other systems, in which such structure has been shown to be required. Here we show, by expressing the HIV-1 gag-pol domain in cultured vertebrate cells, that a stem-loop structure 3' of the HIV-1 shift site is indeed important for wild-type levels of frameshifting in vivo.

Amino Acid Sequence↗
PubMed1993

The MA (p15) and p12 regions of the gag gene are sufficient for the pathogenicity of the murine AIDS virus.

Inoculation of the replication-defective retrovirus DEF27 (BM5d), packaged as an amphotropic virus pseudotype, into C57BL/6J mice leads to development of murine AIDS. Disease development showed a long incubation period (20 to 24 weeks), was associated with amplification of the BM5d provirus in splenocytes and lymph nodes, and was independent of the presence of exogenous or endogenous replication-competent helper viruses. However, both the onset of disease and amplification of the defective provirus were significantly enhanced by coinfection with the replication-competent B-cell-tropic ecotropic helper virus BM5e. The part of the BM5d viral genome that was essential for the pathogenicity was determined by making precisely engineered alterations in the reading frame of the gag and pol genes of BM5d proviral DNA and examining the ability of the altered amphotropic BM5d pseudotypes to induce the disease in C57BL/6J mice. The results show that expression of the MA (p15) and p12 regions of the gag gene is sufficient for pathogenicity of the BM5d retrovirus.

Animals↗
PubMed1995

A single retroviral gag precursor signal peptide recognized by FBL-3 tumor-specific cytotoxic T lymphocytes.

Several dominant T-cell receptors of cytotoxic T-lymphocyte (CTL) clones specific for FBL-3 tumor antigen were clonally amplified in mixed lymphocyte tumor cell cultures derived from an individual immune mouse. Every CTL clone analyzed had a common specificity for a single epitope in the precursor to cell membrane-associated nonstructural gag-encoded protein, Pr75gag, which can be minimally identified by nine amino acid residues, SIVLCCLCL. This epitope is located within the hydrophobic signal sequence motif that mediates translocation of the protein into the endoplasmic reticulum. These novel observations suggest that expression of Pr75gag in FBL-3 tumor cells led to the amplification of CTLs which recognize the signal sequence of the nonstructural gag-encoded glycoprotein precursor.

Amino Acid Sequence↗
PubMed1997

The cytopathicity of a simian immunodeficiency virus Mne variant is determined by mutations in Gag and Env.

Previous studies suggested that the rapidly replicating, highly cytopathic, syncytium-inducing (rapid-high/SI) phenotype of simian immunodeficiency virus Mne variants that evolved in macaques inoculated with a slowly replicating, minimally cytopathic, non-syncytium-inducing (slow-low/NSI) molecular clone was not solely the result of changes in the envelope surface protein (Env SU). To define the viral determinants responsible for the change in phenotype, we molecularly cloned a rapid-high/SI variant (designated SIVMne170) derived from the peripheral blood mononuclear cells (PBMCs) of a pig-tailed macaque that was inoculated with a slow-low/NSI molecular clone, SIVMneCL8. SIVMne170 was SI and replicated with faster kinetics and was more cytopathic than the parent SIVMneCL8 in CEMx174 cells. Additionally, SIVMne170 was more cytopathic for the CD4+ T-cell population than SIVMneCL8 in macaque PBMCs. An analysis of chimeric viruses constructed between the variant SIVMne170 and the parent virus SIVMneCL8 demonstrated that there are determinants encoded within both the 5' and 3' halves of SIVMne170 that independently contribute to its rapid-high/SI phenotype. As we previously observed with other SIVMne variants, the Env SU of SIVMne170 was important for syncytium induction but was not a key determinant of cytopathicity. By contrast, the intracellular domain of the envelope transmembrane protein (Env TM) contributed to both the SI and cytopathic properties of SIVMne170. We also found that the minimal determinant within the 5' half of SIVMne170 that conferred its rapid replication kinetics and cytopathicity mapped to the capsid- and nucleocapsid-encoding regions of gag. Together, these data demonstrate that mutations selected in Gag and Env TM intracytoplasmic tail influence the replication and cytopathicity of SIVMne variants that evolve in the host.

Amino Acid Sequence↗
PubMed1998

Cleavage of human immunodeficiency virus type 1 proteinase from the N-terminally adjacent p6* protein is essential for efficient Gag polyprotein processing and viral infectivity.

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.

Animals↗
PubMed2000

Roles of Pr55(gag) and NCp7 in tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1.

To study in vivo tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR(-)) virus was used as the source of primer tRNA(3)(Lys)/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA(3)(Lys), dCTP, were lower with PR(-) than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR(-) dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55(gag) had no effect on initiation. These results indicate that while Pr55(gag) is sufficient for tRNA(3)(Lys) placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA(3)(Lys) placement and initiation of reverse transcription.

Base Sequence↗
PubMed1999

A threshold level of gag expression is required for particle formation in rat cells transformed by avian retroviruses.

To test the hypothesis that the block of polyprotein precursor processing and particle formation in RSV-transformed mammalian cells is due to a low level of pr76gag expression, rat tumor cell lines with different amounts of precursor molecules were used. The wild-type forms of pr76gag have been expressed at a high level by use of SV40-based vector and thirty-two stable transfected cell clones were isolated. The gag protein expression was detected in the cell lysate by immunoblotting. Untransfected cells released no proteins that could be detected by immunoprecipitation with anti-RSV serum. Membrane-enclosed gag precursor-polyprotein molecules and infectious virus particles from different stably transfected clones have been found in the medium. Both immature and mature virions of type C morphology were directly detected by transmission electron microscopy. Surprisingly, virus-like particles of morphology similar to mature type C retroviruses were found enclosed within intracellular membranes in a stably transfected nonproducing clone.

Animals↗
PubMed1992

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.

Amino Acid Sequence↗
PubMed1991

The HIV-1 gag precursor is processed via two pathways: implications for cytotoxicity.

All retroviruses studied thus far contain proteases which process viral precursors to liberate the structural and enzymatic proteins of the viral capsid. We have examined the processing of the Gag precursor of HIV-1 which is composed of the viral structural proteins. Our results indicate that Gag is processed via two pathways: an expected membrane-associated pathway which gives rise to virions and a cytoplasmic pathway in which processed viral proteins accumulate in the cytoplasm. The presence of an active protease in the cytoplasm of infected cells is a potential source of toxicity. A comparison of the extent of cytoplasmic processing in lytically infected cells compared with that in cells which are not killed by the virus demonstrates a close correlation between cytoplasmic processing and cell killing.

Cell Death↗
PubMed1990

Characterization of clones of HIV-1 infected HuT 78 cells defective in gag gene processing and of SIV clones producing large amounts of envelope glycoprotein.

Single-cell clones of HIV-1 (FRE-3) or SIV/Mne infected HuT 78 cells were obtained by plating dilutions of virally infected HuT 78 cells on a monolayer of sheep choroid plexus cells in 96-well microtiter plates. Several of these clones produce HIV-1 virus mutants that accumulate the gag precursor polyprotein and lack a functional protease. These protease-deficient viruses are non-infectious and consist of aberrant "immature" virus particles as determined by electron microscopy. Several SIV mutants are also described that produce large amounts of either the envelope glycoprotein gp120 or the nucleic acid binding gag protein. These mutants are useful for the purification of these retroviral proteins, in developing assays of protease inhibitors, and in preparing SIV envelope protein vaccines.

Animals↗
PubMed1995

Inhibition of human immunodeficiency virus type 1 (HIV) gag gene expression by an antisense oligodeoxynucleotide phosphorothioate.

We have employed a cos-like monkey kidney cell line (B4.14) transfected with plasmids pCMVgag-pol-rre-r (containing the HIV gag and pol genes) and pCMVrev (containing the HIV rev gene), as a model to investigate whether antisense constructs could interfere with specific HIV gene and protein expression. We utilized an antisense construct (GP12A) directed against a non-regulatory region of the HIV genome, to transfect cells that expressed the above-mentioned HIV genes. Our results show that GP12A was able to attenuate levels of relevant HIV mRNA and gag proteins in the absence of cytotoxic effects.

Animals↗
PubMed1993

HIV experimental vaccines based on the iscom technology using envelope and GAG gene products.

In previous experiments gp160 incorporated into iscom was shown to induce neutralizing antibodies to the homologous as well as the heterologous isolates of HIV-1 (Akerblom et al., AIDS Res., 1991). In the present work we have incorporated into iscoms three defined recombinant DNA products of HIV-1. The carboxy-terminal part of gp120 expressed in E. Coli-PB-1; a chimera containing parts of both p24 and p15 expressed in E. coli-GAG; and baculovirus gp160 cloned in baculovirus and produced in insect cells. Immune responses were induced by the iscom preparations to the homologous antigen as well as to defined recombinant products and to the synthetic peptide RP135 (aa 304-328) harboring a neutralizing epitope. Sera from mice immunized with PB1-iscoms and gp160 (baculo) iscoms were tested in a syncytie inhibition assay. The serum from a mouse immunized with PB1 iscoms reacted strongly with the synthetic peptide RP135 and also neutralized the homologous isolate HIV-1/IIIB with a neutralization titer of 1/64. Three gp160 (baculo) iscom antisera were tested, of which two reacted strongly with the synthetic peptide RP135 but did not neutralize the homologous isolate HIV-1/IIIB. High serum titers were induced in mice by the gp160 iscoms (2 micrograms) to homologous antigen and the recombinant DNA E. coli construct p121 covering part of gp41. The ceilings of the antibody responses were reached after two immunizations. The PB1- and GAG-iscoms required three immunizations to reach the ceiling of the antibody response.

AIDS Vaccines↗
PubMed2000

Analysis of a human immunodeficiency virus type 1 gag mutant with an engineered 110-amino-acid insertion in the matrix protein domain.

A human immunodeficiency virus (HIV) matrix (MA) protein mutant was constructed by duplication of 107 codons of the HIV-1 MA domain. This MA protein duplication mutant (MAII) still could assemble and process particles, had a wild-type (wt) HIV particle density, and possessed reverse transcriptase activity of about 80% of the wild type virus level. The incorporation of HIV Env and viral RNA genome was not greatly affected. The MAII was noninfectious or poorly infectious, however, when pseudotyped with an amphotropic murine leukemia virus envelope protein or with the HIV envelope protein. Although the MAII mutant displayed an immunofluorescence staining pattern similar to that of the wild type virus, subcellular fractionation studies indicated that the membrane association of MAII Gag precursors was unstable under high-salt conditions. Electron microscopic studies showed that the mutant had a decreased density of particle cores compared with that of the wild type virus, suggesting an altered arrangement of the packed proteins. As this insertion in the MA gene caused no major effects on virus assembly implies that the HIV-1 gag has the potential to adapt large insertions of extra coding sequences without loss of the ability to direct particle assembly and release.

Blotting, Western↗
PubMed2006

Immunisation with recombinant modified vaccinia virus Ankara expressing HIV-1 gag in HIV-1-infected subjects stimulates broad functional CD4+ T cell responses.

Virus-specific CD4+ T cells with IL-2-secreting and/or proliferative capacity are detected readily in HIV-1-infected long-term nonprogressors and rarely in persons with untreated progressive infection. The contribution of these cells to viraemia control is uncertain, but this question might be addressed in clinical therapeutic vaccination studies. However, the quality of T helper responses induced by currently available HIV-1 vaccine candidates has not been explored in depth. We determined the effect of vaccination with modified vaccinia virus Ankara (MVA) expressing HIV-1 gag p24/p17 (MVA.HIVA) on HIV-1-specific CD4+ T cell responses in 16 chronically infected, highly active antiretroviral therapy (HAART)-treated subjects using CD8-depleted IFN-gamma ELISPOT assays, intracellular cytokine staining assays for IL-2 and IFN-gamma, and a CFSE-based proliferation assay. Gag-specific CD4+ T cell responses were significantly increased in magnitude and breadth after vaccination and targeted both known and new epitopes, several of which were also recognised by healthy HIV-uninfected volunteers immunised with the same vaccines. The frequencies of CD4+ T cells expressing IL-2 or IFN-gamma, alone or simultaneously, were also augmented. These findings indicate that functional virus-specific T helper cells can be boosted by vaccination in chronic HIV-1 infection. Further evaluation of their role in viraemia control is warranted.

AIDS Vaccines↗
PubMed1996

Relative levels of inhibition of p24 gene expression by different 20-mer antisense oligonucleotide sequences targeting nucleotides + 1129 to + 1268 of the HIV-1 gag genome: an analysis of mechanism.

GPI2A is a 20-mer antisense oligonucleotide sequence that is complementary to a region of the HIV-1 gag gene. An analysis of viral core antigen p24 protein synthesis inhibition was performed with cells expressing HIV-1 proteins, following treatment with GPI2A or eight other unique antisense constructs designed to bind to regions of the gag gene, at positions that 5' or 3' flank the GPI2A target site. GPI2A was found to be the most effective construct, indicating that the GPI2A target region is a particularly sensitive site for antisense activity. An analysis of energy-related parameters important in complementary duplex formation was performed for each antisense construct. Also, the potential of each antisense sequence to exhibit self-complementarity or to self-dimerize was assessed. The results from these analyses provided an explanation for the high specificity and the superior inhibitory characteristics of GPIA when compared to the eight other antisense oligonucleotides. GPI2A exhibited the second most favorable energy-related characteristics for hybridization reactions, and most importantly, unlike the other eight antisense sequences, it did not show the potential to self-complement or to dimerize. The results of this study and a previous investigation of sequence specificity requirements for GPI2A inhibition of HIV-1 gene expression provide strong evidence for an antisense mode of action for this oligonucleotide construct, a useful tool for analysis of viral gene expression and perhaps a potential therapeutic agent.

Animals↗
PubMed1997

Functional domain mapping of HIV-1 Gag proteins.

A series of human immunodeficiency virus type 1 (HIV-1) proviral gag gene mutants carrying bacterial CAT gene were constructed and monitored for the expression of reverse transcriptase and CAT in a highly sensitive single-round replication assay system to determine the defective replication phase in lymphocytic cells. All the mutants displayed no abnormality in the process of transcription and translation at late replication stage. In contrast, some matrix, capsid, and p6 mutants were defective at final phase, that is, assembly and virion release. Most of the mutants including nucleocapsid mutants, which showed normal phenotype at late stage, were defective at early replication phase. From the functional domain map thus obtained, it is evident that HIV-1 Gag proteins are required for both early and late replication phases.

CD4-Positive T-Lymphocytes↗
PubMed1999

Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus.

Retroviruses, such as murine leukemia virus (MuLV), whose gag and pol genes are in the same reading frame but separated by a UAG stop codon, require that 5-10 % of ribosomes decode the UAG as an amino acid and continue translation to synthesize the Gag-Pol fusion polyprotein. A specific pseudoknot located eight nucleotides 3' of the UAG is required for this redefinition of the UAG stop codon. The structural probing and mutagenic analyses presented here provide evidence that loop I of the pseudoknot is one nucleotide, stem II has seven base-pairs, and the nucleotides 3' of stem II are important for function. Stem II is more resistant to single-strand-specific probes than stem I. Sequences upstream of the UAG codon allow formation of two competing structures, a stem-loop and the pseudoknot.

Aldehydes↗