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)

Beside the threat of infection via HIV-containing blood, the ophthalmologist is especially interested in the possibility of HIV infection via the tears of HIV-positive persons. In a first step, we tried to isolate HIV-1 from the peripheral blood lymphocytes (PBL) of 50 HIV-1-antibody-positive persons in different stages of disease and to detect reverse transcriptase (RT) and p24 antigen (p24-Ag) in the supernatant. Simultaneously we carried out the same tests on tears of these patients. In 10 persons tears were collected using Schirmer strips, in 40 persons by means of microcapillaries. In a second step 10 sample pairs (PBL and tears) were tested with the polymerase chain reaction to detect proviral sequences of HIV-1 (gag, pol, env). In the first step it was not possible to isolate HIV-1 from tears, nor was it possible to detect RT or p24-Ag from the supernatant. In contrast, this was successful in 32 of the 50 examined cases for the PBL. In the second step, it was possible to detect gag, pol and env in all 10 PBL samples, while gag and pol could be detected only in one tear sample and env not at all. Our results show that the tears of HIV-positive persons contain extremely low quantities of tissue-infectious units of HIV. In addition, proviral sequences seem to occur in much lower frequency in tears than in PBL. Infection with HIV via tears therefore appears very unlikely. These findings make it possible to assign tears a place in a semiquantitative ranking of different body fluids by HIV-1 concentration.
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PMID:[HIV-1 and tears. Results of virus isolation and polymerase chain reaction (PCR)]. 781 1

Phosphorothioate analogs of oligodeoxynucleotides at a concentration of 2 microM protected Himalayan tahr cells from infection by caprine arthritis encephalitis virus (CAEV) and equine dermis cells from infection by equine infectious anemia virus (EIAV). The characteristics of this inhibition against these lentiviruses are similar to those previously described for the inhibition of HIV-1 in ATH8 cells [17]. Thus, the 28-mer homo-oligomer of cytidine [S-(dC)28] was at least as effective as three anti-sense sequences targeted to the LTR, gag, and env regions of CAEV. The effectiveness of homo-oligomers of equal length was in the order C >> A > T, and a random 28-copolymer with a composition of 2C:1G was as effective as S-(dC)28. Shorter oligonucleotides were less effective (28 > 14 > 5 mers) for all base compositions tested. While replication of a simian type D retrovirus was inhibited by S-(dC)28, this compound did not inhibit the cytopathogenicity of two type C retroviruses, amphotropic murine leukemia virus (MuLV), and baboon endogenous virus, when they were tested in the same cell lines used to support the replication of lentiviruses. Southern blot analysis of the high molecular weight DNA of drug-treated CAEV-infected cells showed that S-(dC)28 was acting at or before the reverse transcription step. Our present data and the earlier finding that S-(dC)28 is a potent in vitro inhibitor of the MuLV reverse transcriptase [15] suggest that S-(dC)28 is acting very early in the replication cycle of these lentiviruses. Since MuLV reverse transcriptase is inhibited in vitro, but its replication is not blocked in permissive cells, our data suggest that the phosphorothioate oligonucleotides are preventing virus attachment.
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PMID:Phosphorothioate oligonucleotides inhibit the replication of lentiviruses and type D retroviruses, but not that of type C retroviruses. 782 17

We investigated permissiveness of the malignantly transformed trophoblast (choriocarcinoma) cell lines JAR, BeWo and JEG-3 to the human T cell lymphotropic virus type I (HTLV-I). After co-culture with the productively infected cell line MT-2 the choriocarcinoma cell lines were analysed for infection over a period of three months. The presence of HTLV-I viral DNA was examined by PCR using primers targeting the gag, pol, env and pX specific sequences. All amplified segments were found consistently in the cell cultures throughout the period of study. Further analysis that aimed to characterize the size variation of the integrated proviral DNA by Southern blotting revealed the presence of integrated proviral sequences which consisted, for the most part, of incomplete genomes. The presence of the full-length HTLV-I genome, however, was unequivocally confirmed in clonally expanded cell cultures derived from the originally infected parental cells. In order to analyse virus expression at the transcriptional level, we used reverse transcriptase (RT)-mediated PCR that was targeted at intra-exon regions (gag, pol, env and pX), and the splicing sites of the env and pX-tax/rex mRNAs. When compared with MT-2 cells, substantially lower levels of all transcripts were found in all the cell lines analysed. We were unsuccessful in attempts to detect viral protein expression using polyvalent or Tax- and Gag-specific monoclonal antibodies by Western blot analysis or immunoprecipitation, and we could not detect any RT activity released into the supernatant of the infected cells either. Collectively, these data suggest that the trophoblastic cells may become persistently but essentially non-productively infected with HTLV-I.
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PMID:Cell-mediated transmission of human T cell lymphotropic virus type I to human malignant trophoblastic cells results in long-term persistent infection. 784 28

An infectious molecular clone of human T-cell leukemia virus type I (HTLV-I) was derived from an HTLV-I-transformed rabbit T-cell line, RH/K30, obtained by coculture of rabbit peripheral blood mononuclear cells (PBMC) with the human HTLV-I-transformed cell line MT-2. The RH/K30 cell line contained two integrated proviruses, an intact HTLV-I genome and an apparently defective provirus with an in-frame stop codon in the env gene. A genomic DNA fragment containing the intact HTLV-I provirus was cloned into bacteriophage lambda (K30 phi) and subcloned into a plasmid vector (K30p). HTLV-I p24gag protein was detected in culture supernatants of human and rabbit T-cell and fibroblast lines transfected with these clones, at levels comparable to those of the parental cell line RH/K30. Persistent expression of virus was observed in one of these lines, RL-5/K30p, for more than 24 months. Biologic characterization of this cell line revealed the presence of integrated HTLV-I provirus, spliced and unspliced mRNA transcripts, and typical extracellular type C retrovirus particles. As expected, these virus particles contained HTLV-I RNA and reverse transcriptase activity. The transfected cells also expressed surface major histocompatibility complex class II, whereas no expression of this molecule was detected in the parental RL-5 cell line. Virus was passaged by cocultivation of irradiated RL-5/K30p cells with either rabbit PBMC or human cord blood mononuclear cells, demonstrating in vitro infectivity. The virus produced in these cells was also infectious in vivo, since rabbits injected with RL-5/K30p cells became productively infected, as evidenced by seroconversion, amplification of HTLV-I-specific sequences by PCR from PBMC DNA, and virus isolation from PBMC. Availability of infectious molecular clones will facilitate functional studies of HTLV-I genes and gene products.
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PMID:Characterization of an infectious molecular clone of human T-cell leukemia virus type I. 788 47

A new subtype A strain of HIV-1, designated HIV-1 IbNg, was isolated from the peripheral blood mononuclear cells (PBMCs) of an HIV-seropositive, but healthy, 23-year-old blood donor from Ibadan, Nigeria. Analysis of the envelope-encoding sequences of HIV-1 strains isolated from the major centers of the AIDS pandemic has identified at least 9 distinct HIV-1 genetic clusters or clades, tentatively designated as A, B, C, D, E, F, G, H, and O. The gp120-coding region of HIV-1 IbNg was characterized. The env gene was amplified from viral RNA using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. RNA was prepared by cocultivations of PBMCs from the Nigerian subject with phytohemagglutinin-stimulated PBMCs from an HIV seronegative donor. The PCR products were cloned into pBluescriptIIsK(+) and sequenced by the dideoxy chain termination method. Nucleotide sequence data were used to generate a consensus nucleotide sequence of the entire gp120-coding region of HIV-l IbNg, using the Sequence Analysis Software Package. Alignment of the nucleotide sequences of the env gene of HIV-1 IbNg with those of other known HIV-1 strains indicated that this virus most closely clustered with HIV-1 strains belonging to the A subtype, which was in agreement with the observation that this subtype is most closely associated geographically with central and western Africa. DNA sequence analysis of the env genes of 4 additional HIV-1 strains (G3, G9, JV1083, and JP88) from Nigeria indicated that these strains clustered most closely with HIV-1 strains belonging to the G subgroup. Characterization of the entire genomic sequence of HIV-1 IbNg is important for identifying molecular structure(s) of viral gene(s) that may be involved in the pathogenesis of subtype A strains in Nigeria. Research is currently underway to obtain a complete sequence of the entire HIV-1 IbNg genome.
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PMID:Sequence analysis of the glycoprotein 120 coding region of a new HIV type 1 subtype A strain (HIV-1IbNg) from Nigeria. 788 38

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.
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PMID:Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation. 798 33

Several previously unnoticed genes in the human immunodeficiency virus type 1 (HIV-1), potentially encoding selenoproteins, have been discovered by analyzing the genomic RNA structure and its relation to novel open reading frames. We have found a number of new potential RNA pseudoknots, including one in the long terminal repeat, several that coincide with highly conserved enzyme active site sequences in the pol coding region, and one in the env coding region. These pseudoknots can potentially direct the synthesis of selenocysteine (SeC) containing--1 frameshift fusion proteins. This is possible because we have found potential SeC insertion sequences (SECIS) in the RNA of HIV and other retroviruses; such structures are known to be necessary and sufficient for the incorporation of SeC at UGA "stop" codons anywhere in a eukaryotic mRNA. In several locations, UGA codons in the -1 reading frame are highly conserved across a broad spectrum of primate immunodeficiency viruses. Due to the degeneracy of the genetic code, this conservation cannot be explained by evolutionary selection of the pol gene protein sequence alone. Such observations, combined with the conservation of the associated reading frames, strongly suggest that these are real genes, and thus that the pseudoknots are also real. A protease pseudoknot-directed -1 frameshift fusion protein contains a highly conserved SeC codon and has significant similarities to a number of DNA binding proteins, including papillomavirus E2 proteins, suggesting it may be a virally encoded repressor of HIV transcription when cleaved by protease from the rest of the gag-pol gene product. A reverse transcriptase (RT) frameshift fusion protein replaces the RT active site with a highly conserved SeC-containing module. An integrase frameshift fusion protein contains the N-terminal integrase DNA-binding domain and a potential ATP-binding "GKS" motif; it has significant similarities to several helicases, but no SeC codons. A potential frameshift fusion protein from env has one SeC codon, but not in a highly conserved position. SeC incorporation could extend the nef gene product by 33 residues through the C-terminal UGA codon without frameshifting, potentially leading to substantial SeC utilization in infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A basis for new approaches to the chemotherapy of AIDS: novel genes in HIV-1 potentially encode selenoproteins expressed by ribosomal frameshifting and termination suppression. 806 94

Two-phase extraction in a system composed of dextran and polyethylene glycol was used to purify simian immunodeficiency virus, SIVMAC251 (32H isolate) from 25 l of culture supernatant. The virus partitioned to the interphase with 80% recovery of gag peptide p27 and reverse transcriptase and an about 25% recovery of the external env glycoprotein, gp148. The virus was treated with octylglycoside and its subcomponents separated. Two gag-p27 containing fractions were obtained; gag-1, which also contained reverse transcriptase and nucleopeptides, and gag-2, which contained the major portion of the p27. The env gp148 was purified by chromatography through a series of lectin columns. The prepared materials are characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immuno- and lectin blotting.
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PMID:Purification of simian immunodeficiency virus, SIVMAC251, and of its external envelope glycoprotein, gp148. 808 61

The env and v-src genes of a duck-adapted variant of Rous sarcoma virus were replaced for corresponding genes from parental chick-derived virus. The generation of viral constructs with replaced genes is described. DNAs of viruses with replaced genes were assayed on chick and duck embryo fibroblasts by transfection assays. Transformation efficiency was measured by the focus assay and multiplication of virus by the reverse transcriptase assay. Duck-adapted virus with replaced env gene lost the higher transformation efficiency for duck cells, whereas replacement of the v-src gene had no effect on its host-specific transformation activity.
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PMID:Role of replaced v-src and env genes in the duck-adapted variant of Rous sarcoma virus. 818 99

Proliferation of the Friend retrovirus was specifically inhibited by the env mRNA complementary oligonucleotide encapsulated in pH-sensitive liposomes. This observation was made using the focus immunoassay (FIA) and the reverse transcriptase test. The key finding of the present study was the dramatic impact on liposome penetration. For chronic or de novo infection, the point at which the penetration of liposomes began corresponded to the time needed for the virus to leave the cell. In the absence of the virus, liposomes remained adsorbed onto the cell surface without any internalization. Regardless of the mechanism involved, the fact that a retroviral infection stimulates the cellular uptake of oligonucleotide liposomes widens the spectrum of strategies for specific antiviral action.
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PMID:Inhibition of the Friend retrovirus by antisense oligonucleotides encapsulated in liposomes: mechanism of action. 827 3


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