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At least 37 records · Page 2Linked to original sources
PubMed1979

Tryptic peptide analysis of gag and gag-pol gene products of Rauscher murine leukemia virus.

[3H]tyrosine-labeled viral precursor polyproteins and known mature viral proteins derived from the Rauscher murine leukemia virus gag and pol genes were examined by two-dimensional tryptic peptide mapping. Pr200gag-pol was found to contain peptide sequences of the viral core proteins p30, p15, p12, and p10, as well as peptide sequences found in the cell-associated reverse transcriptase. Intermediate reverse transcriptase precursor Pr125pol lacked peptide sequences of the four-core proteins but contained reverse transcriptase-specific tryptic peptides plus two additional tyrosine-containing tryptic peptides not related to gag or pol gene products. Methionine-containing tryptic peptide analysis also suggested the presence of additional protein material in Pr125pol (Kopchick et al., Proc. Natl. Acad. Sci. U.S.A. 75:2016-2020, 1978). Pr200gag-pol, although containing both viral core and reverse transcriptase-assoicated methionine and tyrosine tryptic peptides, also contained additional tryptic peptides. Thes are of two classes: (i) tryptic peptides associated with the Pr125pol but not Pr80pol and (ii) tryptic peptides not found in Pr125pol or in any known viral protein. One interpretation of these results is that Pr200gag-pol contains additional gene products aside from the gag and pol genes. Pr80gag and Pr65gag peptide maps were also examined and found to have sequences of all four core proteins. Pr65gag was found to contain two p30 tyrosine tryptic peptides that were absent in Pr80gag, suggesting that Pr80gag may not be the precursor to Pr65gag. Pr80gag, as expected from its larger size, also contained tryptic peptides not found in Pr65gag. Two of these additional Pr80gag tryptic peptides were found in Pr80pol as well but not in any of the viral core proteins, suggesting that Pr80gag and Pr80pol may have overlapping peptide sequences. Consistent with this finding is the conclusion that Pr80gag terminates within the pol gene. A model that describes the relationship of these recent findings to viral gene products is presented.

Amino Acid Sequence↗
PubMed1993

Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I.

For study of the pol gene expression of human T-cell leukemia virus type I (HTLV-I), RNA was transcribed in vitro from proviral DNA and translated in rabbit reticulocyte lysates. This cell-free translation resulted in two major translation products representing the Gag and Gag-Pro polyproteins. By contrast, the Gag-Pro-Pol polyprotein could be readily observed only when translation was performed with mutant mRNA in which the protease (pro) reading frame was aligned to gag to eliminate the frameshifting event in the gag-pro overlap. The results indicated that two independent ribosomal frameshifting events are required for expression of the HTLV-I pol gene product. Studies with mutant DNAs facilitated the characterization of the primary structure of the HTLV-I mRNA responsible for the ribosomal frameshift in the pro-pol overlap and demonstrated that the frameshift occurs at the signal sequence UUUAAAC. Direct amino acid sequencing of the transframe protein localized the site of the frameshift to the asparagine codon AAC.

Amino Acid Sequence↗
PubMed2004

HIV type 1 BF recombinant strains exhibit different pol gene mosaic patterns: descriptive analysis from 284 patients under treatment failure.

Different complex structures of the pol gene have been identified in 284 HIV-1 B/F recombinant sequences obtained from a group of 587 patients under treatment failure from Argentina. To analyze the mosaic structures of these viral sequences and to determine their phylogenetic relationship, the 284 partial pol gene sequences of BF recombinant viruses were amplified by RT-PCR and sequenced. Intersubtype breakpoints were analyzed by bootscanning. Phylogenetic relationships were determined by means of neighbor-joining trees. The analysis of the sequences showed multiple phylogenetic topologies clustering within intersubtype BF reference sequences. At least three different mosaic patterns were found compared to previously described BF-type viruses with unequal distribution in the studied population. The analysis also showed that HIV-1 BF recombinant viruses with diverse mosaic structures are phylogenetically related in their F segments and in selected B fragments with the F1 subtype and with BF recombinant viruses from Brazil, respectively, suggesting a common recombinant ancestor. No association was observed between the prevalence of each mosaic pattern and the frequency of major drug-resistance mutations in PR and RT.

Anti-HIV Agents↗
PubMed2004

Peptide insertions in reverse transcriptase pol gene of human immunodeficiency virus type 1 as a rare cause of persistent antiretroviral therapeutic failure.

Peptide insertions in codons 67-71 of the reverse transcriptase (RT) pol gene were detected in 11 (2.7%) of 414 genotypic analyses performed in a hospital cohort of 2900 outpatients with human immunodeficiency virus type 1 (HIV-1) infection. The duration of antiretroviral treatment (bi- or tri-therapy) before the detection of insertions ranged from 12 to 60 months. Dipeptide insertions were detected in ten patients, of which the most frequent was serine-serine. A monopeptide insertion was diagnosed once. The amino-acid composition patterns of insertions varied with time in five of the 11 patients. Peptide insertions were always associated with various patterns of pre-existing or appearing resistance mutations in the RT pol gene to different antiretroviral drugs. Genotypic-guided treatment resulted in virological and immunological improvement in two patients. In contrast, the remaining patients did not respond to any of the various antiretroviral regimens prescribed. Furthermore, various patterns of resistance mutations developed to the prescribed antiretroviral drugs, with AIDS-related conditions leading to death in two patients. It was concluded that peptide insertion in this region of the HIV-1 RT pol gene constitutes a rare cause of persistent therapeutic failure, and that management of such patients remains challenging despite successive genotypic analyses aimed at detecting mutations conferring antiretroviral drug resistance.

Adult↗
PubMed1979

Tryptic peptide analysis of avian oncovirus gag and pol gene products.

Radiolabeled tryptic peptides of the gag and pol gene products of avian oncoviruses were examined. This analysis included Rous-associated virus 2 structural proteins and the Pr76gag and P180gag-pol proteins in Rous-associated virus 2-infected chicken embryo cells. The methionine- and cysteine-containing tryptic peptides of virion internal structural proteins were present in both Pr76gag and P180gag-pol, suggesting that there was no loss of gag gene-coding sequences during the generation of P180gag-pol. No overlap of gag and pol gene structural information was detected. Analysis of intermediates in the processing of Pr76gag and translation inhibition mapping with pactamycin yielded the following order of structural proteins within the Rous-associated virus 2 Pr76gag precursor: NH2-p19-p12-p27-p15-COOH. The gag and pol sequences missing in the endogenous gsmp120 protein of uninfected gs+ chicken cells were identified by comparison with those of Rous-associated virus 2 P180gag-pol.

Animals↗
PubMed1991

Identification of a binding site in the hepatitis B virus RNA pregenome for the viral Pol gene product.

The hepatitis B virus, although containing a DNA genome, replicates by reverse transcription of an RNA pregenome. The viral Pol gene encodes the reverse transcriptase which catalyzes viral DNA synthesis. To study the interaction of this protein with HBV RNA, the entire Pol gene product was expressed except its eight amino-terminal codons in Escherichia coli as fusion protein with beta-galactosidase. In the absence of competing nucleic acids full-length expression products were able to nonspecifically bind in vitro synthesized HBV RNAs of different polarity and length. However, if competed with an excess of unspecific RNA, only those HBV RNAs were bound which contained besides the direct repeats 1 and 2 nucleotide sequences downstream of direct repeat 1. The corresponding binding site was found to be located within the adjacent 134 nucleotides downstream of DR1. We conclude from our data that this region which is in part homologous to the U5 region of retroviral genomes may be important for the binding of the HBV Pol gene product to the viral pregenome.

Binding Sites↗
PubMed1984

The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus.

We mutagenized cloned spleen necrosis virus DNA to identify a region of the retrovirus genome encoding a polypeptide required for integration of viral DNA. Five plasmids bearing different lesions in the 3' end of the pol gene were examined for the ability to integrate or replicate following transfection of chicken embryo fibroblasts. Transfection with one of these DNAs resulted in the generation of mutant virus incapable of integrating but able to replicate at low levels; this phenotype is identical to that of mutants bearing alterations in the cis-acting region, att. To determine whether the 3' end of the pol gene encodes a protein that interacts with att, we did a complementation experiment. Cells were first infected with an att- virus and then superinfected with the integration-deficient virus containing a lesion in the pol gene and a wild-type att site. The results showed that the att- virus provided a transacting function allowing integration of viral DNA derived from the mutant bearing a wild-type att site. Thus, the 3' end of the pol gene serves as an "int" locus and encodes a protein mediating integration of retrovirus DNA through interaction with att.

Animals↗
PubMed1995

Impaired infectivity of HIV-1 after a single point mutation in the POL gene to escape the effect of a protease inhibitor in vitro.

To examine whether the mutation of protease in an HIV-1 resistant to a protease inhibitor affects the virus phenotype in vitro, the infectivity of the protease inhibitor-escape-virus was compared to that of the parent virus. In different T-cell lines, the infectivity of the escape virus was impaired by 10-fold compared to the parent virus. MT-4 cell killing by the escape virus, measured using the MTT assay, was much weaker than that by the parent virus. The escape virus contained more unprocessed Pr55gag than the parent virus. A delayed appearance of mature p24 in cells chronically infected with the escape virus was also noticed by the pulse-chase method. The same findings were obtained using pNL432 (HIV-1 DNA molecular clone) with the same mutation in the protease gene. Despite the lack of a significant difference in virus binding, less unintegrated and integrated DNA was detected in MT-4 cells infected with the escape virus compared to the parent virus. The impaired infectivity of the escape virus may be explained by the inefficient maturation of Gag proteins, due to the mutated protease, which may affect an early step in the virus life cycle.

Antigens, CD↗
PubMed1999

Mutations designed to alter the stability of a putative RNA duplex in the HIV-1 pol gene.

In the HIV-1 integrase coding region there is a polypurine tract (PPT) involved in the initiation of provirus plus-strand synthesis. Upstream of this PPT there is a 15-nucleotide inverted repeat (IR) complementary to most of the PPT. We have constructed one mutant with five amino acid-neutral U to C and A to G changes in the IR and one mutant with corresponding amino acid-neutral changes in the PPT. Each set of changes abolished the complementarity and suppressed the replication of HIV-1 slightly. The combination of these ten changes restored the complementarity, and doubled the calculated free energy of the putative duplex between the IR and the PPT. This double mutant did not replicate under normal conditions, possibly because the reverse transcriptase was unable to penetrate the duplex. However, when high loads of the double mutant were added to permissive cells, replicative HIV did occasionally appear. The resurrected virus harvested from these cells replicated consistently, even though the ten nucleotide changes were left unchanged. There were no compensatory mutations in the vicinity of the IR/PPT or in the reverse transcriptase gene.

Animals↗
PubMed1997

Mutations in the pol gene of human immunodeficiency virus type 1 in infected patients receiving didanosine and hydroxyurea combination therapy.

The pattern of mutations in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) strains that confer resistance to didanosine (ddI) was analyzed in 2 groups of patients receiving either ddI monotherapy or ddI plus hydroxyurea (HU) combination therapy. Twelve patients receiving combination therapy and 8 receiving monotherapy were tested. Combinations of ddI plus HU did not prevent the onset of mutations, which emerged in 50% of the patients in this group compared with 25% of the ddI monotherapy group. In addition, in 1 patient from the combination therapy arm, who had a limited response to the therapy, an unusual pattern of mutations was found: the insertion of 2 amino acids between residues 69 and 70, a region critical for resistance to nucleoside analogs. The higher efficacy of the combination of HU and ddI compared with that of ddI monotherapy cannot be attributed to a delayed or decreased onset of resistance to ddI.

Anti-HIV Agents↗
PubMedDate not supplied

Viral dynamics and mutations in the pol gene during primary HIV-1 infection.

The understanding of viral dynamics and appearance of mutations during primary infection could be useful for the design of an efficient therapy. For this reason a cohort of samples from naive primary patients was examined. The results pointed out that only a few secondary mutations in protease gene (having no effect on resistance) were found, while a single mutation conferring resistance to non-nucleosides inhibitors of reverse transcriptase was found both in plasma and cerebrospinal fluid of a patient. As both the protease secondary mutations and the single non nucleoside reverse transcriptase mutation map far from the catalytical sites of the enzymes, neither one is able to impair viral fitness. Overall data suggest that treated donors carrying resistant strains may be in part unable to transfer them to the recipient, and/or virus in the recipient tends to revert to wild type. These results should be taken into account in the planning of early HAART treatment of HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors.

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

5' Untranslated Regions↗