Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene.
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PMID:Generation and characterization of the human immunodeficiency virus type 1 mutants. 170 90

The prevalence of simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus type 1 (STLV-I), and type D retrovirus (SRV-D) antibodies was determined for 1229 rhesus monkeys (Macaca mulatta) from two research colonies. Serum samples were tested by using enzyme-linked immunosorbent assay (ELISA), immunoblot (IB), and radioimmunoprecipitation assay (RIPA). Seropositive results for the three retroviruses tested were 0 for SIV, 270 (22%) for STLV-I, and 103 (8.4%) for type D retrovirus. Of the rhesus monkey sera, 61 (5.0%) were reactive to SIV gag p27 only, when tested by IB, but were negative when further tested by RIPA. Virus isolation was attempted from cultured peripheral blood mononuclear cells of 35 monkeys whose sera contained only p27 reactivity and none were positive by reverse transcriptase and core antigen assays to detect SIV. No overt clinical signs of immunodeficiency disease or unexplained deaths were evident in either monkey colony. Additionally, 63 of 165 (38%) human sera from various groups (primate center workers, normal donors, health care workers) had weak to moderate IB reactivity only to SIV p27, but 31 of 31 sera tested were negative by RIPA. These sera remained reactive to SIV p27 following absorption with an uninfected cell lysate, after blocking IB strips with various blocking solutions and were reactive to different SIV antigen preparations while remaining negative to human immunodeficiency virus type 1 (HIV-1) by IB and negative to HIV-2 by ELISA. These data underscore the need to adopt criteria for a positive SIV serologic test requiring reactivity against more than one viral gene product. These results also illustrate a potential problem in the testing of human sera for antibodies against simian retroviruses and demonstrate the need for caution in the interpretation of immunoblot results.
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PMID:SIV, STLV-I and type D retrovirus antibodies in captive rhesus macaques and immunoblot reactivity to SIV p27 in human and rhesus monkey sera. 170 6

The time course of viral gene expression in H9 cells acutely infected with HIV-1 was analysed by the polymerase chain reaction (PCR). Virus-specific sequences were first detected in genomic DNA of H9 cells 1-2 h after infection. RNA for the regulatory genes such as the tat and nef appeared 2-3 h post-infection and RNA for the gag and env at 3 h. The results demonstrate that viral DNA synthesis occurs rapidly after infection of target cells followed by synthesis of viral RNA. Cell-associated reverse transcriptase activity increased after 24 h, while culture supernatant enzyme activity increased later, between 1-5 days. The delay in virus release after rapid integration and transcriptional activity suggests the involvement of additional factors, perhaps both cellular and viral, that control the formation and budding of mature virions.
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PMID:Kinetics of early HIV-1 gene expression in infected H9 cells assessed by PCR. 170 54

Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27-pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity.
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PMID:Identification and characterization of human immunodeficiency virus type 1 gag-pol fusion protein in transfected mammalian cells. 170 86

Human immunodeficiency virus (HIV) IIIB expression and Epstein-Barr virus (EBV) B95.8-induced transformation were studied during coinfection. Coinfection of peripheral blood lymphocyte (PBL) cultures with HIV and EBV resulted in down-regulation of HIV expression. EBV-induced and spontaneous transformation were markedly reduced in PBL cultures exposed to HIV before EBV. On the other hand, transformation was enhanced when PBL cultures were infected with HIV either simultaneous to or after EBV. Reconstitution of EBV-infected B cell cultures with autochthonous T cells demonstrated that HIV-infected T cells had a reduced ability to inhibit EBV-induced transformation. PHA stimulation of HIV-infected T cells eliminated their ability to inhibit EBV-induced transformation. Lymphoblastoid cell lines (LCLs) established from coinfected PBLs expressed B cell markers and were EBV positive, while a large proportion of the LCLs expressed HIV antigens, released reverse transcriptase activity into the supernatant, and produced syncytia when cocultivated with indicator cell line SupT1. HIV provirus could be detected in LCLs established from coinfected cultures by PCR amplification using specific sets of amplimers for gag and env genes of HIV. To more closely examine the role of various cell types in lymphocyte transformation and HIV replication during coinfection, experiments were carried out using subpopulations enriched for either B or T cells. Simultaneous coinfection of purified B cells with EBV and HIV resulted in a marked reduction of HIV expression, whereas EBV-induced transformation was enhanced. In contrast, spontaneous B cell transformation was inhibited by HIV. A proportion of LCLs established from purified B cells coinfected with EBV and HIV expressed HIV antigens, released reverse transcriptase activity, and produced syncytia on SupT1 cells. These results demonstrate that the IIIB strain of HIV and B95.8 strain of EBV can interact during coinfection of B cells to alter the course of virus expression.
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PMID:Altered HIV expression and EBV-induced transformation in coinfected PBLs and PBL subpopulations. 170 29

To generate nonpathogenic viral particles which express active human immunodeficiency virus type 1 (HIV-1) protease (PR), plasmids containing sequences from the genomes of HIV-1 and Moloney murine leukemia virus (M-MuLV) were constructed. Either the PR coding region alone; the gag, PR, and reverse transcriptase protein-coding regions; or the complete gag and pol protein-coding regions from HIV-1 were substituted for the corresponding regions of a full-length M-MuLV clone to yield the chimeric plasmids pMoHIV-I, pMoHIV-III, and pMoHIV-IV, respectively. Cell lines which express the viral gag polyprotein were isolated for hybrids pMoHIV-I and pMoHIV-III. These cells produced viral particles which contained processed core proteins. Cleavage of the gag polyprotein in the viral particles was inhibited by the HIV-1 PR inhibitor L-687908, indicating that the viral PR is responsible for the observed processing. The hybrid virions were not infectious; analyses indicated that the viral particles contained little or no reverse transcriptase activity. In addition, particles produced by pMoHIV-III transfectants failed to package the viral genomic RNA. The cell line which expresses and processes the HIV-1 gag polyprotein is a safe and effective reagent for the in vivo evaluation of potential inhibitors of the HIV-1 PR.
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PMID:Expression of active human immunodeficiency virus type 1 protease by noninfectious chimeric virus particles. 170 93

Intracisternal A particles (IAPs) are retrovirus-like structures consistently observable in a variety of mouse tumor cells such as myeloma and hybridoma and in early embryonic cells derived from rodents but nothing is known of their infectivity. Mouse IAPs contain a gag-like protein, a reverse transcriptase and a polyadenylated RNA molecule (IAP RNA). DNA sequences complementary to IAP RNA (IAP genome) are interspersedly present in rodent such as mice, rats, Chinese hamsters and Syrian hamsters at several hundred to a thousand copies per haploid genome. Molecularly cloned IAP genomes from two species Mus and Syrian hamster were 6 to 8 kb in length with LTRs of about 0.4 kb long. The nucleotide sequence of the Syrian hamster IAP genome, H18, predicted a typical LTR-gag-prt-pol-env-LTR structure, although many stop codons were present in the region corresponding to env. The comparison of the deduced amino acid sequences of the pol region showed IAP (type A), mouse mammary tumor virus (MMTV) (type B), and squirrel monkey retrovirus (SMRV) (type D) genomes to be closely related. By using a DNA fragment encoding the pol region of the Syrian hamster IAP genome, human endogenous retroviruses termed HERV-K, were cloned from a fetal human liver gene library. Typical HERV-K genome was 9.5 kb in length having LTRs of about 1.0 kb. The HERV-K provirus could encode gag (666 codons), prt (334 codons), pol (937 codons), and env (618 codons) genes. HERV-K was shown to be closely related to types A, B and D retroviruses. The HERV-K genomes are present at about 50 copies per haploid human genome. In several human tumor cell lines, the HERV-K genome was expressed as 8.8 kb poly(A)+ RNA which appeared to be a full-size transcript of this genome. In the human breast cancer cell line T47D, stimulation of HERV-K genome expression was observed following female steroids treatment. In a detailed investigation on the organization of HERV-K proviruses in human genome, we found repetitive sequences homologous to the LTR region of the HERV-K genome. They were about 630 bp in length with an A rich tail at 3' end and found to be a SINE type nonviral retroposon. These elements were present at 4,000 to 5,000 copies per haploid human genome.
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PMID:Molecular biology of type A endogenous retrovirus. 171 Jun 82

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA primer bound near the 5' end of the genomic RNA at a position termed the primer-binding site (PBS). The PBS is an 18-nucleotide region of the HIV-1 genome complementary to cellular tRNA(Lys). To further investigate the sequence requirements for the PBS in reverse transcription, deletions in the PBS were created and subcloned into a plasmid containing the infectious HIV-1 proviral genome. The mutations deleted the entire PBS (delta PBS) or the first 9 (delta 1-9), the second 9 (delta 10-18), or 12 (delta 7-18) nucleotides of the PBS. An additional mutation in the PBS was created in which the second nine nucleotides were deleted and nine additional nucleotides were substituted [Lys(1-9)]. The transfection of plasmids containing the wild-type or mutant proviral genomes into tissue culture cells resulted in expression of the HIV-1 gag and env gene products, as determined by immunoprecipitation using sera from AIDS patients. HIV-1 virus was released from the transfected cells, as determined by analysis of the supernatants for reverse transcriptase activity. The infectivity of the viruses derived from the transfection was examined by coculture experiments with SupT1 cells, which support high-level replication of HIV-1. The transfection of plasmids containing HIV-1 proviral genomes with the delta PBS and PBS (delta 1-9) mutations did not produce infectious virus. In contrast, the HIV-1 proviral genomes with the delta 10-18, delta 7-18, and Lys(1-9) mutations in the PBS produced infectious virus upon transfection, although the kinetics of appearance was significantly delayed for the mutant viruses compared with the wild type. To further explore the nature of this defect, the PBS region from integrated proviral genomes was amplified by polymerase chain reaction and individual DNA products were subcloned into M13mp19, followed by a sequence analysis of the PBS region from individual M13 phage clones. In each of the PBS regions examined, the 18-nucleotide PBS complementary to tRNA(Lys) was present. However, nucleotide deletions and insertions were found 3' to the PBS from the samples derived from the transfection of plasmids containing mutant proviral genomes. Upon reinfection, the revertant viruses maintained the deletions 3' to the PBS and had kinetics of replication similar to that of the wild-type virus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription. 171 13

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

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We described previously the construction of PR-defective avian leukosis viruses. These mutant viruses are noninfectious, and their major internal components are the uncleaved gag and gag-pol polyproteins (Pr76gag and Pr180gag-pol). The reverse transcriptase (RT) activity associated with the PR-defective virions is approximately 500-fold reduced relative to that of wild-type virions, suggesting that specific cleavages activate RT activity. To gain a better understanding of the role that PR plays in the processing and activation of RT, we performed complementation experiments wherein wild-type or PR mutant gag precursors were separately coexpressed with frame-corrected wild-type or PR mutant gag-pol precursors. The results demonstrate that, as in other retrovirus systems, gag-pol precursors can be assembled into virions only when they are rescued by a gag precursor. If the gag precursor is wild type, then the rescued Pr180gag-pol is completely and properly matured, irrespective of whether its embedded PR domain is wild type or mutant. In both cases, the virions produced are fully and equally infectious. This indicates that an active-site mutation in the PR domain of the gag-pol precursor has no effect on avian leukosis virus infectivity when particles are assembled from wild-type gag precursors. In contrast, if the gag precursor has an active-site mutation in PR or is deleted for PR, then the virions are noninfectious and the gag and gag-pol precursors remain unprocessed, even if the embedded PR domain of Pr180gag-pol is wild type. Thus, in this system, virion-associated Pr180gag-pol displays no detectable cis- or trans-acting PR activity. As assayed with an exogenous template, virions with processed gag-pol polyprotein display high levels of RT activity while those with unprocessed Pr180gag-pol display greatly reduced RT activity. These results demonstrate that during virion assembly, the PR supplied by a gag precursor is both necessary and sufficient for trans-activation of RT through proteolytic maturation of copackaged gag-pol polyprotein.
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PMID:trans-acting viral protease is necessary and sufficient for activation of avian leukosis virus reverse transcriptase. 171 19


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