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At least 19 recordsLinked to original sources

PubMed1995

Phylogenetic analyses of 55 retroelements on the basis of the nucleotide and product amino acid sequences of the pol gene.

Comparisons of pol gene nucleotide and reverse transcriptase (RT) amino acid sequences of 47 retroviruses, 3 caulimoviruses, and 5 hepadnaviruses showed that approximately one-third of the gene at the 5' end is much more conserved than other pol regions. The most conserved regions on both the nucleotide and amino acid sequences were chosen for construction of phylogenetic trees. The maximum-parsimony and distance-matrix methods were used for analyses of aligned amino acid sequences; these two methods, and the compatibility method, were used to analyze the aligned nucleotide sequences. Essentially identical majority-rule consensus trees were produced by these different methods from both the pol gene nucleotide and RT amino acid sequences, which divided the 55 retroelements into six major groups. The reliability of the phylogenetic trees was probed with the bootstrapping of 100 replicates of the original sequence alignments. The grouping results were shown to be statistically significant by multiple comparisons with the least-significant-difference procedure.

Amino Acid Sequence↗

PubMed1987

Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain.

Three pol gene products have been identified in avian retroviral particles: the full-length 95-kilodalton (kDa) beta chain of reverse transcriptase and two proteolytic cleavage products of beta, a 63-kDa reverse transcriptase alpha chain derived from the amino terminus of beta and a 32-kDa (pp32) endonuclease from its carboxy terminus. By using molecularly cloned retroviral DNA and synthetic oligonucleotides to introduce initiator ATGs and codons corresponding to the authentic N termini, we constructed two bacterial-expression clones; one clone contains the entire pol gene, and the other contains the region encoding the pp32 domain. A 99-kDa protein was synthesized in Escherichia coli by the full-length clone, and a 36-kDa protein was synthesized by the endonuclease domain clone. The recombinant proteins exceeded the size of both the mature viral beta chain and the pp32, respectively, by approximately 4 kDa. These larger sizes, however, are consistent with predictions from the DNA sequence of the pol gene. Processing of the recombinant pol proteins was examined by using p15 protease purified from virus particles and antisera directed against synthetic peptides corresponding to three domains in pol. Proteolytic digestion of the 99-kDa product with p15 produced a 63-kDa protein that comigrated on polyacrylamide gels with the alpha chain of reverse transciptase and a 36-kDa fragment that comigrated with the endonuclease domain product. Further digestion of the 36-kDa protein yielded a 32-kDa protein that comigrated with viral pp32 endonuclease. Thus, we concluded that two p15-sensitive sites exist in pol. Cleavage at the previously identified site produces alpha, and cleavage at the newly discovered site removes approximately 4 kDa from the C terminus of the primary protein product. Since the 36-kDa protein was also detected in protein isolated from virus particles, it seems probable that processing at the C-terminal site is a normal step in the production of mature beta and pp32 endonuclease products.

Amino Acid Sequence↗

PubMed1991

Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.

Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity.

Animals↗

PubMed1996

The human foamy virus pol gene is expressed as a Pro-Pol polyprotein and not as a Gag-Pol fusion protein.

It has been reported recently that the human foamy virus (HFV) Pol polyprotein of 120 kDa is synthesized in the absence of the active HFV aspartic protease. To gain more information on how the 120-kDa Pro-Pol protein is synthesized, mutant HFV genomes were constructed and the resulting proviruses were analyzed with respect to HFV pol expression and infectivity. HFV proviruses that contain termination codons in the nucleocapsid domain of gag and thus lack a gag-pol overlap region assumed to be required for translational frameshifting, nevertheless expressed the 120-kDa Pro-Pol precursor, the 80-kDa reverse transcriptase/RNase H, and a 40-kDa integrase in amounts similar to those observed for wild-type genomes. Since a Gag-independent expression of authentic Pol proteins was detectable in cells transfected with eukaryotic HFV pol expression plasmids, the data indicate that the HFV Pol precursor of 120 kDa is expressed independently of Gag by a mechanism that does not rely on ribosomal frameshifting, since the postulated HFV Gag-Pol protein of 190 kDa was not detectable under the conditions used. Furthermore, replacement of the Met residue by Thr at position 9 in pol within the gag-pol overlap region resulted in strongly reduced HFV Pol polyprotein expression and infectivity of the resulting proviruses. This Met residue of pol conserved in foamy virus sequences is the likely candidate for translational initiation of the 120-kDa Pro-Pol polyprotein. trans complementation of the HFV mutant with the Met-to-Thr substitution in the pol gene by a eukaryotic plasmid that expressed the HFV Pro-Pol protein resulted in partial recovery of infectivity. When HFV pol was fused in frame to gag, an engineered 190-kDa Gag-Pol fusion protein was formed and the enzymatic activity of the HFV protease was partially retained. The results imply that HFV is the first retrovirus that expresses a Pol polyprotein without formation of a Gag-Pol fusion protein.

Amino Acid Sequence↗

PubMed1985

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation.

Animals↗

PubMed1991

Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study.

An ultrastructural study was performed on rabbit epithelial RK-13 cells and CD4+ human T lymphocyte lines infected with various recombinant vaccinia viruses (RVVs) expressing genes of human immunodeficiency virus (HIV): the mature p17 or p24 gag domain alone, the entire or truncated gag gene, the reverse transcriptase domain, or the gag-pol genes with a frameshift mutation. Cells infected with RVVs that produced the gag polyprotein with a predicted Mr of more than 48K showed budding and release of HIV-like particles into the extracellular space. These particles were not observed in cells expressing a truncated gag gene (p17 and p24 regions). Mature HIV-like particles were observed extracellularly when the entire gag gene and the protease region of the pol gene were expressed. In contrast, in cells infected with RVVs that contained the gag-pol gene with a frameshift mutation, neither recognizable budding structures nor extracellular HIV-like particles could be detected. These results suggest that the gag gene, particularly its 3' terminus, is necessary for the assembly of HIV particles. In addition, the protease region of the pol gene seems to be required for morphological maturation of HIV particles, but complete proteolytic cleavage of the gag protein may prevent bud formation.

Amino Acid Sequence↗

PubMed1990

Relationship of the env genes and the endonuclease domain of the pol genes of simian foamy virus type 1 and human foamy virus.

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related.

Amino Acid Sequence↗

PubMed1984

Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection.

We have used in vitro mutagenesis to explore the functions of the gene products encoded by the pol gene of Moloney murine leukemia virus (M-MuLV). Deletions were constructed at a variety of positions in the gene, and the altered DNA copies of the viral genome were introduced into mouse cells by cotransformation. The mutants could be divided into two classes depending on the phenotype and map position of the deletion within the pol gene. Mutants with deletions mapping in the 5' portion of the gene were found to be completely deficient in reverse transcriptase activity. Mutants mapping in the 3' portion of the gene, however, assembled and released virions with normal levels of reverse transcriptase and RNAase H activities. When applied to permissive cells, these virions directed the synthesis of all three forms of unintegrated viral DNA: full-length, double-stranded linear DNA and the two circular forms with one and two copies of the long terminal repeat sequences. The infection was arrested at this point and the infected cells did not become producers of virus. Thus the 3' portion of the pol gene encodes a polypeptide with a function distinct from that of reverse transcriptase, which is not required for synthesis of viral DNA but is essential for establishment of that DNA in a stable, active form in the infected cell. We suggest that this function may be the integration of the proviral DNA.

Chromosome Deletion↗

PubMed1986

Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes.

A computer analysis of the amino acid sequences from the putative gene products of retroviral pol genes has revealed a 150-residue segment that is homologous with the ribonuclease H of Escherichia coli. The segment occurs at the carboxyl terminus of the region assigned to the 90-kDa reverse transcriptase polypeptide. In contrast, a section nearer the amino terminus of this sequence can be aligned with nonretroviral polymerases. The order of activities in the pol gene is thus: polymerase-ribonuclease-endonuclease. On another note, all retroviral endonuclease sequences contain a consensus zinc-binding "finger." This should not be confused with the well-known zinc requirement of reverse transcriptases.

Amino Acid Sequence↗

PubMed1986

Murine mammary tumor virus pol-related sequences in human DNA: characterization and sequence comparison with the complete murine mammary tumor virus pol gene.

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.

Amino Acid Sequence↗

PubMed1991

Evidence that less-than-full-length pol gene products are functional in hepadnavirus DNA synthesis.

Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase domains had a leaky phenotype and, depending on the location and type of mutation, synthesized up to 10% as much viral DNA as did the wild type. This region of the pol gene had previously been reported to be refractory to missense mutations; in fact, the leakiness of most of our mutants appeared attributable to translational suppression, which would also be expected to introduce amino acid changes. However, at least one mutant (pH1093 + 2), which was ca. 10% as active as the wild type, appeared to use a novel pathway to express the viral pol gene. Our analyses indicated that pH1093 + 2 synthesized the viral reverse transcriptase as a fusion protein with the amino-terminal portion of the pre-S envelope protein. Thus, in this case, the products of the terminal-protein and reverse transcriptase domains of the pol gene would function as separate protein species, though perhaps noncovalently joined in a dimeric structure during assembly of DNA replication complexes. Evidence was also obtained that was consistent with the idea that the wild-type pol gene may, at least in certain instances, be expressed as functional, subgenic polypeptides.

Base Sequence↗

PubMed1997

Development of pathological lesions in the central nervous system of transgenic mice expressing the env gene of ts1 Moloney murine leukemia virus in the absence of the viral gag and pol genes and viral replication.

The ts1 Moloney murine leukemia virus causes a degenerative neurologic disease in mice characterized by the development of noninflammatory spongiform encephalomyelopathy. To determine whether gag and pol gene products and viral replication are necessary for the ts1-env gene product to cause neurodegeneration, we generated transgenic mice harboring only ts1-env. Neuropathological lesions were observed in mice expressing the transgene in the central nervous system. This implies that gag and pol gene products and viral replication are not necessary for ts1-env to cause a mild form of neurodegeneration in mice.

Animals↗

PubMed1985

Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting.

The pol gene of Rous sarcoma virus is positioned downstream of the gag gene in a different, briefly overlapping reading frame; nevertheless, the primary translation product of pol is a gag-pol fusion protein. Two mechanisms, ribosomal frameshifting and RNA splicing, have been considered to explain this phenomenon. The frameshifting model is supported by synthesis of both gag protein and gag-pol fusion protein in a cell-free mammalian translation system programmed by a single RNA species that was synthesized from cloned viral DNA with a bacteriophage RNA polymerase. Under these conditions, the ratio of the gag protein to the fusion protein (about 20 to 1) is similar to that previously observed in infected cells, the frameshifting is specific for the gag-pol junction, and it is unaffected by large deletions in gag. In addition, synthesis of the fusion protein is ten times less efficient in an Escherichia coli cell-free translation system and cannot be explained by transcriptional errors or in vitro modification of the RNA. Ribosomal frameshifting may affect production of other proteins in higher eukaryotes, including proteins encoded by several retroviruses and transposable elements.

Animals↗

PubMed1984

Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure.

The molecular defect in the nonconditional B-tropic MuLV pol mutant, clone 23 (Gerwin et al., J. Virol. 31:741-751, 1979), has been characterized by recombinant DNA technology. The entire mutant genome was cloned from an EcoRI digest of integrated cellular DNA into bacteriophage lambda Charon 4A and then subcloned at the EcoRI site of pBR322. NIH-3T3 cells transfected with the plasmid clone, termed pRTM (RTM, reverse transcriptase mutant), reproduced the properties of clone 23 virus-infected cells. In vivo ligation experiments involving cotransfection of subclones of pRTM and wild-type murine leukemia virus localized the defect in the clone 23 genome to an approximately 400-base-pair region in the pol gene between the SalI and XhoI sites. Sequence analysis of this region in the wild-type and mutant genomes revealed that the mutant has one additional C residue located 231 bases downstream of the last base of the SalI recognition site. This 1-base insertion brings three TGA termination codons into phase. Thus, the mutation in clone 23 leads to premature termination of translation, explaining the presence in clone 23 virions of a truncated polymerase with low levels of enzymatic activity. It was previously shown that the gag precursor is cleaved normally in clone 23-infected cells; therefore, if a virus-coded protease is involved in this cleavage, it must be encoded by sequences upstream of the reverse transcriptase region of the pol gene. This consideration, coupled with the observed molecular weight of the mutant polymerase and our precise determination of its C terminus, have led to a proposal for the genetic organization of the murine leukemia virus pol gene.

Animals↗

PubMed1992

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient.

Acquired Immunodeficiency Syndrome↗

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↗

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↗