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)

An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) non-lymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAGM LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.
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PMID:Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone. 131 47

We have isolated a bovine prolactin (bPRL) receptor cDNA from an endometrial cDNA library, which predicts a 557 amino acid transmembrane protein similar to the long forms of other characterized prolactin receptors. The predicted cytoplasmic domain is slightly truncated primarily by a stop codon located 36 codons 5' from the stop utilized in the human hepatic transcript. When expressed in COS cells, this cDNA was shown to encode a protein which bound bovine placental lactogen (bPL) and bPRL with nearly equal affinity (KD for bPL, 2.03 x 10(-10) M; bPRL, 3.07 x 10(-10) M). Northern analysis demonstrated multiple transcripts, with maternal liver, corpus luteum, intestine, endometrium and fetal liver containing a major transcript of about 3.8 kb, and maternal corpus luteum and endometrium, a second sized transcript of apparently equal abundance of 4.4 kb. This difference did not appear to be within the coding region. Primer extension analysis of maternal hepatic and endometrial transcripts revealed considerable heterogeneity. Examination of the distribution of prolactin and growth hormone receptor transcripts at mid-pregnancy by semi-quantitative reverse transcriptase polymerase chain reaction showed that both are widespread in bovine fetal and placental tissues. This isolation of bovine prolactin receptor cDNA, and description of receptor distribution will facilitate study of the action of the placental and pituitary members of this gene family during pregnancy.
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PMID:Molecular cloning of the bovine prolactin receptor and distribution of prolactin and growth hormone receptor transcripts in fetal and utero-placental tissues. 133 25

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

Low molecular weight RNA in HIV-1 is found in three size classes resembling 7S RNA, 5S RNA, and tRNA. The 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) patterns of tRNA found in HIV-1 have been determined in virus produced in five different cell types: H9, UHC1 (a U937-derived clone), UHC8 (an RT(-) derivative of U937), HeLa, and COS. The presence of the putative primer tRNA for reverse transcriptase, tRNA(Lys,3), has also been determined either by hybridization with a tRNA(Lys,3)-specific DNA probe or by a comparison of the electrophoretic mobility of viral tRNA species with purified human tRNA(Lys,3). Our results indicate the following: 1) The number of tRNA species found in infectious HIV-1IIIB produced in different cell types varies, according to cell type, from greater than 20 to 4, indicating that only 4 or less tRNA species are required for the viral infectious life cycle. 2) There are 1-3 tRNA species tightly associated to the viral genomic RNA, depending upon the cell type producing the virus. 3) The putative primer tRNA, tRNA(Lys,3), is detected with the tRNA(Lys,3)-specific hybridization probe in the tRNA of HIV-1 produced in H9 cells, and the tightly associated tRNA species in this virus has the same electrophoretic mobility in 1-D PAGE as purified tRNA(Lys,3). On the other hand, we cannot detect tRNA(Lys,3) in the tRNA of HIV-1 produced in HeLa cells, and the tightly associated tRNA found in this virus does not migrate with the same electrophoretic mobility as tRNA(Lys,3).
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PMID:Variable tRNA content in HIV-1IIIB. 162 25

Unlike many other reverse transcriptase genes, the polymerase (P) gene of the hepatitis B viruses is expressed by translational initiation from its own AUG codon rather than by ribosomal frameshifting during translation of the overlapping core gene (C). To explore the mechanism of its translation, we have fused the P gene of duck hepatitis B virus to the bacterial lacZ gene at a point 3' to the C-P overlap; this allows detection of the products of P translation with antisera to the lacZ-encoded protein. The C and P/Z coding regions were cloned downstream of a heterologous promoter and expressed in COS-7 cells. A single, bicistronic mRNA containing both C and P sequences is detected in these cells, and translational initiation occurs efficiently at the internally situated P AUG. Mutations affecting C translation have only minimal effects on P expression, in contrast to what would be expected from a modified scanning model for translation. We conclude that P translation depends on a mechanism other than scanning to allow internal entry of ribosomes to the region of the P initiator.
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PMID:Mechanism of translation of the hepadnaviral polymerase (P) gene. 169 11

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

Deletions were constructed within a functional human immunodeficiency virus type 1 (HIV-1) proviral clone in order to assess the role of the envelope protein in virus particle formation. A graded exonuclease deletion technique was used to produce 12 clones with deletions of 175-308 nucleotides in the first conserved domain of envelope. This included 9 clones with frameshift deletions and 3 clones with in-frame deletions. Isogenic pairs of env deletion clones were produced with or without an additional deletion in the vif and vpr genes. Upon transfection, all clones produced virus particles, as determined by p24 antigen, reverse transcriptase, and sucrose gradient assays with conditioned media. Virus particles produced from clones with deletions in env or vif and vpr, or both regions, banded on sucrose gradients with a mobility similar to that of virus produced by the parental clone. The p24 gag capsid protein in the particles was resistant to trypsin, but the particles were disrupted by treatment with Triton X-100, suggesting the presence of a surrounding lipid bilayer. Furthermore, electron microscopic studies revealed both mature and immature virus particles derived from COS cells transfected with the env deletion clones. Cocultivation experiments with lymphoid cells and cells transfected with each of the env deletion clones demonstrated that the virus particles were noninfectious.
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PMID:Formation of noninfectious HIV-1 virus particles lacking a full-length envelope protein. 182 17

The expression of the gag-pol polyprotein of human immunodeficiency virus type 1 (HIV-1) occurs via ribosomal frameshifting between the gag and pol genes. Because low levels of the gag-pol precursor are naturally produced in HIV-1-infected cells, a limited amount of information is available on the biology of this molecule. To further study this polyprotein, two mutant HIV-1 proviral genomes were created to position the gag and pol genes in the same translational reading frame. The mutations inserted a single thymidine nucleotide at the site of ribosomal frameshifting (nucleotide 1635), which results in the addition of a phenylalanine residue (frameshift 1 [FS1]), or a single adenine nucleotide, which results in the addition of a leucine residue (frameshift 2 [FS2]). Transfection of the mutant proviral genomes into COS-1 cells resulted in the expression of the p160gag-pol polyprotein precursor as well as the proteolytically processed gag and pol gene products. Metabolic labeling of the transfected cells with [3H]myristic acid revealed that the p160gag-pol and p17gag proteins expressed from the mutant genomes were myristylated. While the supernatants from COS-1 cells transfected with wild-type or mutant proviral genomes contained similar amounts of p24 antigen, the levels of reverse transcriptase were, on the average, 10 times greater in the supernatants from cells transfected with the FS1 and FS2 proviral genomes. The cells transfected with the wild-type proviral genome released infectious viral particles, while the mutant proviral genomes released p24 and reverse transcriptase in the absence of detectable particle formation. The mutant proviral genomes were completely noninfectious as determined by coculture of the transfected COS-1 cells with SupT1 cells. These results demonstrate that the gag-pol polyprotein of HIV-1 contains the appropriate signals for proteolytic processing and association with intracytoplasmic membranes in the absence of virion formation.
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PMID:Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production. 187 Feb 15

Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.
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PMID:Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. 215 62

It is generally believed that the gag gene product of human immunodeficiency virus type 1 (HIV-1) is processed into several core proteins by a virus-specific protease. We used deletion mutation analysis to study the role of HIV-specific protease in the processing of core proteins and its requirement for viral infectivity. Several mutant genomes with deletions in the protease gene were constructed. A mammalian cell line, COS-M6, transfected with the wild-type viral genome was shown to produce virions containing processed core proteins, while COS-M6 cells transfected with two mutated genomes could express only the core protein precursor, Pr56gag. The wild-type transfectant produced infectious virus; both transfectants expressing the mutated genomes also produced virions, and one of them still retained reverse transcriptase activity. However, the mutant viral particles were devoid of infectivity. Virions with a distinct central core and an electron-dense nucleoid budded out from the plasma membrane of COS-M6 cells transfected with the wild-type genome. In contrast, noninfectious virions that budded either into cytoplasmic vacuoles or out from the plasma membrane of COS-M6 cells transfected with mutant genomes contained ring-shaped nucleoids. These results indicate that the HIV-1 protease plays a role not only in the maturation of the core proteins but also in the assembly of the virus and thus is required for viral infectivity.
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PMID:Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. 265 99


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