Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The expression of the pol gene of human immunodeficiency virus type 1 occurs via a ribosomal frameshift between the gag and pol genes. The resulting protein, a Gag-Pol polyprotein, is produced at a level 5 to 10% of that of the Gag protein. The Gag-Pol polyprotein is incorporated into virions and provides viral protease, reverse transcriptase, and integrase, which are essential for infectivity. It is generally believed that the Gag-Pol polyprotein is incorporated into virions via interaction with the Gag protein, although the details of the mechanism are unknown. To further study this problem, we have constructed a human immunodeficiency virus type 1 proviral genome which overexpresses the Gag-Pol polyprotein (Pr160gag-pol). Transfection of this proviral genome (pGPpr-) into COS-1 cells resulted in the expression of full-length Pr160gag-pol polyprotein. Although the majority of the Pr160gag-pol was confined to the cells, low levels of reverse transcriptase activity were detectable in the cell supernatants. The cotransfection of pGPpr- with a second plasmid which expresses only the Pr55gag precursor (pGAG) resulted in a significantly higher level of Pr160gag-pol in the medium of transfected cells. Sedimentation analysis using sucrose density gradients demonstrated that most Pr160gag-pol was found in fractions corresponding to the density of virion particles, indicating that the Pr160gag-pol polyprotein was released in association with a Pr55gag viruslike particle. To further characterize the requirements for the release, a mutation was constructed to express an unmyristylated Pr160gag-pol polyprotein. Coexpression with Pr55gag demonstrated that the unmyristylated Pr160gag-pol was also incorporated into virion particles. Subcellular fractionation experiments revealed that the distributions of the Pr160gag-polmyr- and Pr160gag-pol in the membrane and cytosol were similar under low- or high-ionic-strength conditions. Taken together, these results suggest that myristylation of the Pr160gag-pol polyprotein is not required for the interaction with the Pr55gag necessary for packaging into a viruslike particle.
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PMID:The nonmyristylated Pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with Pr55gag and is incorporated into viruslike particles. 138 61

The retroviruses of the avian sarcoma-leukosis virus group synthesize their viral protease (PR) in two precursor forms--as a carboxy-terminal domain of the Gag precursor and as an embedded domain within the Gag-Pol precursor. We have shown previously that the Gag-derived PR is fully capable of processing the Gag precursor in the absence of the embedded PR (R.P. Bennett, S. Rhee, R.C. Craven, E. Hunter, and J.W. Wills, J. Virol. 65:272-280, 1991). In this study, we examined the question of whether or not the PR domain of Gag-Pol has an essential role in the maturation of the Pol proteins. The Gag-Pol precursor was expressed in the absence of Gag by use of a simian virus 40-based vector in which the gag and pol reading frames were fused. The fusion protein accumulated to high levels in transfected cells without being released into the medium but could be rescued into particles by coexpression of the Gag protein from a second vector. The resulting particles contained mature Gag and Pol proteins and active reverse transcriptase (RT). Using this complementation system, the effects of PR defects in the Gag and/or Gag-Pol proteins on the activation of RT were examined. The results showed that the presence of a functional PR on the Gag precursor, but not on Gag-Pol, was required for full activation of RT. The embedded PR of Gag-Pol was unable to carry out any detectable processing of the Gag precursor and was able to activate RT to only a low level in the absence of a functional Gag PR domain. Finally, some point mutations in the Gag-Pol PR domain inhibited activation of RT in trans by a wild-type PR, suggesting that the correct conformation of the PR domain in Gag-Pol is prerequisite for activation of RT.
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PMID:Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly. 171 18

We investigated the production of Gag particles by Vero, CV-1, or 1D cells infected with different vaccinia virus recombinants expressing HIV gag or gag-pol genes. Immunoblots of (centrifuged) culture media from 1D cells infected with vMM5, a vaccinia virus recombinant expressing the HIV-2 gag-pol genes, revealed the presence of abundant particles that contained (mostly processed) Gag antigens. In contrast, Gag particles were found only in low amounts in the culture medium from Vero cells infected with the same HIV gag-pol vaccinia virus recombinant; the Gag precursor remained associated with the infected Vero cells and was efficiently processed. This low excretion of Gag particles after infection of Vero cells with vMM5 was also demonstrated by assays of reverse transcriptase activity in the pellet of centrifuged culture medium. Cell fractionation showed that Gag proteins were predominantly found in the membrane fraction from both 1D and Vero cells. Electron microscopy observations of 1D or of Vero cells infected with vMM5 vaccinia virus recombinant revealed in both cases the presence of particles budding at the plasma membrane. However, the shape of the budding particles was different in the two cell lines, with immature forms present in the membrane from the infected Vero cells. An inefficient excretion of Gag particles was also observed after infection of Vero cells with different vaccinia virus recombinants expressing either an uncleaved HIV-2 Gag protein or the HIV-1 gag-pol genes, as judged both by immunoblot and reverse transcriptase activity assays.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cell line-dependent release of HIV-like gag particles after infection of mammalian cells with recombinant vaccinia viruses. 752 Jul 22

Walleye dermal sarcoma virus (WDSV) is a fish retrovirus associated with the development of tumors in walleyes. We have determined the complete nucleotide sequence of a DNA clone of WDSV, the N-terminal amino acid sequences of the major proteins, and the start site for transcription. The long terminal repeat is 590 bp in length, with the U3 region containing consensus sequences likely to be involved in viral gene expression. A predicted histidyl-tRNA binding site is located 3 nucleotides distal to the 3' end of the long terminal repeat. Virus particles purified by isopycnic sedimentation followed by rate zonal sedimentation showed major polypeptides with molecular sizes of 90, 25, 20, 14, and 10 kDa. N-terminal sequencing of these allowed unambiguous assignment of the small polypeptides as products of the gag gene, including CA and NC, and the large polypeptide as the TM product of env. The 582-amino-acid (aa) Gag protein precursor is predicted to be myristylated as is found for most retroviruses. NC contains a single Cys-His motif like those found in all retroviruses except spumaviruses. The WDSV pro and pol genes are in the same translational reading frame as gag and thus apparently are translated after termination suppression. The env gene encodes a surface (SU) protein of 469 aa predicted to be highly glycosylated and a large transmembrane (TM) protein of 754 aa. The sequence of TM is unusual in that it ends in a very hydrophobic segment of 65 residues containing a single charged residue. Following the env gene are two nonoverlapping long open reading frames of 290 aa (orf-A) and 306 aa (orf-B), neither of which shows significant sequence similarity with known genes. A third open reading frame of 119 aa (orf-C) is located in the leader region preceding gag. The predicted amino acid sequence of reverse transcriptase would place WDSV phylogenetically closest to the murine leukemia virus-related genus of retroviruses. However, other members of this genus do not have accessory genes, suggesting that WDSV acquired orf-A, orf-B, and perhaps orf-C late in its evolution. We hypothesize by analogy with other complex retroviruses that the accessory genes of WDSV function in the regulation of transcription and in RNA processing and also in the induction of walleye dermal sarcoma.
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PMID:Nucleotide sequence and protein analysis of a complex piscine retrovirus, walleye dermal sarcoma virus. 763 75

We studied the effects of gag mutations on human immunodeficiency virus type 1 (HIV-1) assembly, processing, and infectivity by using a replication-defective HIV expression system. HIV mutants were screened for infectivity by transduction of a selectable marker and were examined for assembly by monitoring particle release from transfected cells. Gag protein processing and reverse transcriptase activities of mutant particles were also assayed. Surprisingly, most Gag protein mutants were assembled and processed. The two exceptions to this rule were a myristylation-minus mutant, and one gag matrix domain mutant which expressed proteins that were trapped intracellularly. Interestingly, a mutant with a 56-amino-acid deletion within the HIV gag capsid domain still could assemble and process virus particles, exhibited a wild-type retrovirus particle density, and had wild-type reverse transcriptase activity. Indeed, although most HIV-1 gag mutants were noninfectious or poorly infectious, they produced apparently normal particles which possessed significant reverse transcriptase activities. These results strongly support the notion that the HIV-1 Gag proteins are functionally involved in post-assembly, postprocessing stages of virus infectivity.
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PMID:Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants. 768 14

SDZ NIM 811 is a cyclosporin A analog that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. The mechanism of action of SDZ NIM 811 is clearly different from those of all other anti-HIV agents described so far. In cell-free assays, it is not an inhibitor of reverse transcriptase, protease, integrase, and it does not interfere with Rev or Tat function. SDZ NIM 811 does not down-regulate CD4 or inhibit fusion between infected and uninfected, CD4-expressing cells. p24 production from chronically HIV-infected cells is not impaired either. To elucidate the mode of action of SDZ NIM 811, we performed DNA PCR analysis in HIV-1 IIIB-infected MT4 cells in one cycle of virus replication. The effects of SDZ NIM 811 on the kinetics of viral DNA synthesis, appearance of two-long terminal repeat circles (2-LTR circles), and integration of DNA were studied. SDZ NIM 811 inhibited 2-LTR circle formation in a concentration-dependent manner, which is indicative of nuclear localization of preintegration complexes. Half-maximal inhibition was achieved at 0.17 microgram/ml; this concentration is close to the 50% inhibitory concentrations (0.01 to 0.2 microgram/ml) for viral growth inhibition. As expected, integration of proviral DNA into cellular DNA was also inhibited by SDZ NIM 811. Analysis of the viral particles produced by SDZ NIM 811-treated, chronically infected cells revealed amounts of capsid proteins, reverse transcriptase activity, and viral RNA comparable to those of the untreated control. However, these particles showed a dose-dependent reduction in infectivity (50% inhibitory concentration of 0.028 microgram/ml) which indicates that the assembly process is also impaired by SDZ NIM 811. Gag proteins are postulated to play a role not only in assembly but also in early steps of viral replication, e.g., nuclear localization of the preintegration complex. Recently, it was reported that HIV-1 Gag protein binds to cyclophilin A, the intracellular receptor for cyclosporin A. Interference with Gag-cyclophilin interaction may be the molecular basis for the antiviral activity of cyclosporin A and its analogs.
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PMID:Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus type 1 (HIV-1): interference with early and late events in HIV-1 replication. 781 48

The cis- and trans-acting components of the Rev regulatory pathway employed by equine infectious anemia virus (EIAV) to regulate and coordinate viral gene expression were examined in complementation experiments. Viral protein expression and mRNA expression were compared in cells transiently transfected with wild-type or mutant proviruses in combination with Rev expression plasmids. Mutation of the predicted rev gene abolished Gag protein synthesis, and this defect was complemented, in trans, by Rev. Analysis of viral mRNAs from transfected cells confirmed that EIAV expresses five major mRNAs: the full-length and singly spliced mRNAs contain introns and encode viral structural proteins while the three fully spliced mRNAs, encoding nonstructural genes, are generated by alternative splicing. Compared to cells transfected with the wild-type provirus, the intron-containing mRNAs produced from the rev-minus mutant were present at reduced levels in the nuclear RNA fraction and were not detected in the cytoplasm. This pattern of viral mRNA synthesis was restored to the wild-type pattern by providing Rev in trans. In contrast to the intron-containing mRNAs, cytoplasmic accumulation of the multiply spliced class of mRNAs was independent of Rev. Closer examination of the multiply spliced class of viral mRNAs by reverse transcriptase-PCR analysis revealed a Rev-dependent alternative splicing phenomenon. In the absence of Rev, proviruses expressed a four-exon mRNA at high levels; the addition of Rev caused both a decrease in the levels of the four-exon mRNA and the appearance of a related mRNA lacking exon 3. The cis-acting RNA elements that mediate Rev responsiveness were studied with deleted proviruses, which revealed that EIAV contains at least two elements located near the ends of envelope gene. Unlike the Rev-responsive elements in other retroviruses, the cis-acting regions of EIAV do not appear to form complex secondary structures.
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PMID:Equine infectious anemia virus trans-regulatory protein Rev controls viral mRNA stability, accumulation, and alternative splicing. 815 75

A mutant of Rous sarcoma virus was constructed in which the nine amino acids that separate the CA and NC sequences in the Gag protein were deleted. The spacer peptide deletion mutant produced particles containing the normal complement of viral RNA and all of the viral proteins, including reverse transcriptase. Though electron microscopy revealed particles of normal morphology, the particles were noninfectious. The normally slow maturation of the CA protein, which involves cleavage of the spacer peptide from the carboxy terminus, was bypassed in this mutant, and the association between CA and the internal components of the core appears to have been disrupted. The results suggest that the spacer peptide has an essential role in directing folding and/or oligomerization of the CA subunits within the capsid structure.
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PMID:Necessity of the spacer peptide between CA and NC in the Rous sarcoma virus gag protein. 839 79

The expression of the pol gene of human immunodeficiency virus type 1 (HIV-1) occurs by a ribosomal frameshift between the gag and the pol genes. The Gag-Pol polyprotein is produced at levels of 5 to 10% of that of the Gag protein, and is incorporated into virions to provide the viral protease, reverse transcriptase, and integrase which are essential for replication. The mechanism(s) by which the Gag-Pol polyprotein are targeted to the HIV virion is unknown, although it is believed to be via an interaction with the Gag protein. To further explore the mechanism by which the Gag-Pol polyprotein is incorporated into virions, we have constructed a mutation which changes an aspartic acid in the protease active site to asparagine (pHXB2pro-); a four-amino-acid insertion into the protease gene (pHXB2Smal); and insertion of translational termination codons in the protease gene following the gag gene (pHXB55). Transfection of these proviral genomes into COS-1 cells resulted in intracellular expression of only Pr55gag, demonstrating the inactivation of the viral protease. The expression of Pr55gag was evident in cells transfected with pHXB2pro- during a short pulse and first 3 hr of chase period, whereas at later times the intracellular levels of Pr55gag were greatly reduced. In contrast, the intracellular Pr55gag expressed from transfection of pHXB2Smal or pHXB55 were evident even after 6- or 12-hr chase times. To ascertain the effects of the mutations on the assembly and release of viruslike particles, the supernatants from the transfected cells were analyzed for the presence of Pr55gag. The release of Pr55gag from cells transfected with pHXB2pro- occurred as early as 1 hr following chase period, and increased for up to 3 hr. In contrast, reduced levels of Pr55gag were detected in the medium from cells transfected with pHXB2Smal or pHXB55. Subcellular fractionation studies demonstrated that the Pr55gag expressed from transfection of pHXB2pro- was rapidly targeted to intracellular membranes, while the majority of the Pr55gag expressed from transfection of pHXB2Smal or pHXB55 was distributed evenly between the cytoplasm and membrane fractions. Finally, the released viruslike particles obtained from the transfection of proviral genome pHXB2pro- were stable to mild detergent treatment, whereas particles obtained from transfection of pHXB2Smal and pHXB55 were relatively unstable. These results demonstrate that subtle changes in the Gag-Pol polyprotein of HIV-1 can have significant effects on the assembly and physical stability of the released virus.
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PMID:Mutations in the protease gene of human immunodeficiency virus type 1 affect release and stability of virus particles. 850 89

The capsid domain (CA) of the retroviral Gag protein is a major constituent of the virion core. To examine the role of this protein in M-MuLV morphogenesis and replication, a series of substitution mutations affecting the central region of CA were introduced into an infectious proviral DNA. The altered DNAs were introduced into cells, and the resulting lines were analyzed for production of infectious virions. Only one of the replication defective mutants analyzed was blocked in virion assembly. The remaining mutant DNAs induced the formation and release of particles containing genomic RNA and polymerase protein. The reverse transcriptase associated with these mutant virions was capable of transcribing both minus strand strong stop and extended DNA products using the endogenous genomic RNA as template in vitro. Upon infection of fresh cells, however, no viral DNA synthesis could be detected either by Southern analysis or by an RNase protection assay developed specifically to detect intermediate products of reverse transcription. The results indicate that the bulk of the CA mutants are blocked before reverse transcription of the viral genome and suggest an important role for the capsid protein in an early stage of viral replication.
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PMID:Amino acid substitutions in the CA protein of Moloney murine leukemia virus that block early events in infection. 880 18


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