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Query: UNIPROT:P23193 (transcription elongation factor)
 739 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymes and factors, required for in vitro transcription of templates regulated by vaccinia virus intermediate stage promoters, are present in HeLa cells infected with vaccinia virus in the presence of an inhibitor of DNA replication. Previous studies indicated that in vitro transcription could be reconstituted by adding a partially purified transcription factor to the viral RNA polymerase and capping enzyme. By using an independent purification procedure, we isolated two vaccinia virus intermediate were necessary for transcription of several different intermediate stage promoter templates but not for early or late stage promoter templates. VITF-1 was purified to homogeneity, and the sequences of two tryptic peptides were mapped to the fourth open reading frame within the HindIII E fragment (E4L) of the vaccinia virus genome, which had previously been shown to encode an RNA polymerase subunit of 30 kDa (RPO30) with homology to eukaryotic transcription elongation factor SII. Co-chromatography of VITF-1 with the E4L-derived protein was demonstrated using specific antiserum. In addition, transcriptionally active recombinant VITF-1 was made by expressing the E4L open reading frame in Escherichia coli. Thus, E4L encodes a multifunctional protein, serving as a RNA polymerase subunit and a stage-specific transcription factor. The stepwise binding of capping enzyme, VITF-1, and VITF-2 to a DNA/viral RNA polymerase complex was demonstrated.
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PMID:Purification and identification of a vaccinia virus-encoded intermediate stage promoter-specific transcription factor that has homology to eukaryotic transcription factor SII (TFIIS) and an additional role as a viral RNA polymerase subunit. 818 10

We developed a system to identify the viral proteins required for the packaging and passage of human respiratory syncytial virus (RSV) by reconstructing these events with cDNA-encoded components. Plasmids encoding individual RSV proteins, each under the control of a T7 promoter, were cotransfected in various combinations together with a plasmid containing a minigenome into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Supernatants from these cells were passaged onto fresh cells which were then superinfected with RSV. Functional reconstitution of RSV-specific packaging and passage was detected by expression of the reporter gene carried on the minigenome. As expected, the four nucleocapsid proteins N, P, L, and M2-1 failed to direct packaging and passage of the minigenome. Passage was achieved by further addition of plasmids expressing three membrane-associated proteins, M, G, and F; inclusion of the fourth envelope- associated protein, SH, did not alter passage efficiency. Passage was reduced 10- to 20-fold by omission of G and was abrogated by omission of either M or F. Coexpression of the nonstructural NS1 or NS2 protein had little effect on packaging and passage except through indirect effects on RNA synthesis in the initial transfection. The M2-1 transcription elongation factor was not required for the generation of passage-competent particles. However, addition of increasing quantities of M2-1 to the transfection mediated a dose-dependent inhibition of passage which was alleviated by coexpression of the putative negative regulatory factor M2-2. Omission of the L plasmid reduced passage 10- to 20-fold, most likely due to reduced availability of encapsidated minigenomes for packaging. However, the residual level of passage indicated that neither L protein nor the process of RSV-specific RNA synthesis is required for the production and passage of particles. Omission of N or P from the transfection abrogated passage. Thus, the minimum RSV protein requirements for packaging and passaging a minigenome are N, P, M, and F, although the efficiency is greatly increased by addition of L and G.
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PMID:Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles. 962 Oct 29

Prior genetic analysis suggests that there may exist an interaction between the products of the vaccinia virus genes A18R, a putative negative transcription elongation factor, and G2R, a putative positive transcription elongation factor. In addition, affinity purification of polyhistidine-tagged G2R protein overexpressed in vaccinia virus-infected cells, reported here, results in copurification of the vaccinia H5R protein, previously characterized as a late viral transcription factor. We have therefore used several methods to screen further for interactions among the G2R, A18R, and H5R proteins. Methods include copurification or co-immunoprecipitation of proteins overexpressed during vaccinia virus infection, activation of the gal 4 promoter by gal 4 fusions in the yeast two-hybrid system, and co-immunoprecipitation of proteins synthesized in vitro in a rabbit reticulocyte lysate. The results reveal interactions which include all possible pairwise combinations of the three proteins G2R, A18R, and H5R; however, not all possible permutations of the interactions are observed and the interactions are not observed in all environments tested. The results suggest that the vaccinia virus proteins G2R, A18R, and H5R interact as part of a higher order transcription complex.
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PMID:Characterization of the interactions among vaccinia virus transcription factors G2R, A18R, and H5R. 963 70

Loss of vaccinia virus A18R gene function results in an aberrant transcription profile termed promiscuous transcription, defined as transcription within regions of the genome which are normally transcriptionally silent late during infection. Promiscuous transcription results in an increase in the intracellular concentration of double-stranded RNA, which in turn results in activation of the cellular 2-5A pathway and subsequent RNase L-catalyzed degradation of viral and cellular RNAs. One of three hypotheses could account for promiscuous transcription: (i) reactivation of early promoters late during infection, (ii) random transcription initiation, (iii) readthrough transcription from upstream promoters. Transcriptional analysis of several viral genes, presented here, argues strongly against the first two hypotheses. We have tested the readthrough hypothesis by conducting a detailed transcriptional analysis of a region of the vaccinia virus genome which contains three early genes (M1L, M2L, and K1L) positioned directly downstream of the intermediate gene, K2L. The results show that mutation of the A18R gene results in increased readthrough transcription of the M1L gene originating from the K2L intermediate promoter. A18R mutant infection of RNase L knockout mouse fibroblast (KO3) cells does not result in 2-5A pathway activation, yet the virus mutant is defective in late viral gene expression and remains temperature sensitive. These results demonstrate that the A18R gene product is a negative transcription elongation factor for postreplicative viral genes.
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PMID:The vaccinia virus A18R DNA helicase is a postreplicative negative transcription elongation factor. 969 93

Prior phenotypic analysis of a vaccinia virus gene A18R mutant, Cts23, showed the synthesis of longer than wild type (Wt) length viral transcripts during the intermediate stage of infection, indicating that the A18R protein may act as a negative transcription elongation factor. The purpose of the work described here was to determine a biochemical activity for the A18R protein. Pulse-labeled transcription complexes established from intermediate virus promoters on bead-bound DNA templates were assayed for transcript release during an elongation step that contained nucleotides and various proteins. Pulse-labeled transcription complexes elongated in the presence of only nucleotides were unable to release nascent RNA. The addition of Wt extract during the elongation phase resulted in release of the nascent transcript, indicating that additional factors present in the Wt extract are capable of inducing transcript release. Extract from Cts23 or mock-infected cells was unable to induce release. The lack of release upon addition of Cts23 extract suggests that A18R is involved in release of nascent RNA. By itself, purified polyhistidine-tagged A18R protein (His-A18R) was unable to induce release; however, release did occur in the presence of purified His-A18R protein plus extract from either Cts23 or mock-infected cells. These data taken together indicate that A18R is necessary but not sufficient for release of nascent transcripts. We have also demonstrated that the combination of A18R protein and mock extract induces transcript release in an ATP-dependent manner, consistent with the fact that the A18R protein is an ATP-dependent helicase. Further analysis revealed that the release activity is not restricted to a vaccinia intermediate promoter but is observed using pulse-labeled transcription complexes initiated from all three viral gene class promoters. Therefore, we conclude that A18R and an as yet unidentified cellular factor(s) are required for the in vitro release of nascent RNA from a vaccinia virus transcription elongation complex.
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PMID:Vaccinia virus gene A18R DNA helicase is a transcript release factor. 1062 2

Vaccinia virus genes A18 and G2 affect the elongation and termination of postreplicative viral gene transcription in opposite ways. Viruses with mutations in gene A18 produce abnormally long transcripts, indicating that A18 is a negative transcription elongation factor. Viruses containing mutations in gene G2 produce transcripts that are abnormally short, truncated specifically from their 3' ends, indicating that G2 is a positive transcription elongation factor. Despite the fact that both A18 and G2 are essential genes, A18-G2 double-mutant viruses are viable, presumably because the effects of the mutations are mutually compensatory. In addition, the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) seems to enhance elongation during a vaccinia infection: IBT treatment of a wildtype vaccinia infection induces a phenotype identical to an A18 mutant infection, and G2 mutant viruses are dependent on IBT for growth, presumably because IBT restores the G2 mutant truncated transcripts to a normal length. These observations inspire two independent genetic selections that have now been used to identify an additional vaccinia gene, J3, that regulates postreplicative transcription elongation. In the first selection, a single virus that contains an extragenic suppressor of the A18 temperature-sensitive mutant, Cts23, was isolated. In the second selection, several spontaneous IBT-dependent (IBT(d)) mutant viruses were isolated and characterized genetically. Marker rescue mapping and DNA sequence analysis show that the extragenic suppressor of Cts23 contains a point mutation in the J3 gene, while each of seven new IBT(d) mutants contains null mutations in the J3 gene. The J3 protein has previously been identified as a (nucleoside-2'-O-)-methyltransferase and as a processivity subunit for the heterodimeric viral poly(A) polymerase. The nature of the two independent selections used to isolate the J3 mutants strongly suggests that the J3 protein serves as a positive postreplicative transcription elongation factor during a normal virus infection.
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PMID:The vaccinia virus bifunctional gene J3 (nucleoside-2'-O-)-methyltransferase and poly(A) polymerase stimulatory factor is implicated as a positive transcription elongation factor by two genetic approaches. 1075 13

Prior genetic analysis suggests that the vaccinia virus J3 gene product, previously characterized as a bifunctional (nucleoside-2'-O-)-methyltransferase and poly(A) polymerase stimulatory factor, is a postreplicative positive transcription elongation factor. To test this hypothesis, viruses bearing mutations in the J3 gene were characterized with respect to viral protein and RNA synthesis in infected cells. The analysis reveals that compared to wt virus infections, J3 mutants synthesize reduced amounts of large late viral proteins and shorter-than-normal intermediate and late mRNAs. Structural analysis of one late mRNA shows that it is specifically truncated from the 3' end, thus accounting for its shorter than normal chain length. Thus J3 mutant viruses are defective in elongation of transcription of postreplicative viral genes, strongly suggesting that the J3 gene product normally acts as a positive transcription elongation factor. Biochemical analysis of one J3 missense mutant demonstrates that it retains poly(A) stimulatory activity but is defective in (nucleoside-2'-O-)-methyltransferase activity. Thus the elongation factor activity of the J3 gene product is independent of the poly(A) stimulatory activity. It remains to be determined whether the (nucleoside-2'-O-)-methyltransferase and elongation factor activities of the J3 protein are linked or can be uncoupled by mutation.
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PMID:Transcription elongation activity of the vaccinia virus J3 protein in vivo is independent of poly(A) polymerase stimulation. 1075 14

J3R, the 39-kDa subunit of vaccinia virus poly(A) polymerase, is a multifunctional protein that catalyzes (nucleoside-2'-O-)-methyltransferase activity, serves as a poly(A) polymerase stimulatory factor, and acts as a postreplicative positive transcription elongation factor. Prior results support an association between poly(A) polymerase and the virion RNA polymerase. A possible direct interaction between J3R and H4L subunit of virion RNA polymerase was evaluated. J3R was shown to specifically bind to H4L amino acids 235-256, C terminal to NPH I binding site on H4L. H4L binds to the C-terminal region of J3R between amino acids 169 and 333. The presence of a J3R binding site near to the NPH I binding region on H4L led us to evaluate a physical interaction between NPH I and J3R. The NPH I binding site was located on J3R between amino acids 169 and 249, and J3R was shown to bind to NPH I between amino acids 457 and 524. To evaluate a role for J3R in early gene mRNA synthesis, transcription termination, and/or release, a transcription-competent extract prepared from cells infected with mutant virus lacking J3R, J3-7. Analysis of transcription activity demonstrated that J3R is not required for early mRNA synthesis and is not an essential factor in early gene transcription termination or transcript release in vitro. J3R interaction with NPH I and H4L may serve as a docking site for J3R on the virion RNA polymerase, linking transcription to mRNA cap formation and poly(A) addition.
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PMID:Interaction between the J3R subunit of vaccinia virus poly(A) polymerase and the H4L subunit of the viral RNA polymerase. 1116 28

Previous genetic and biochemical experiments have shown that the vaccinia virus J3 protein has three different roles in mRNA synthesis and modification. First, J3 is a (nucleoside-2'-O-)methyltransferase which methylates the 2' position of the first transcribed nucleotide, thus converting a cap-0 to a cap-1 structure at the 5' ends of mRNAs. Second, J3 is a processivity factor for the virus coded poly(A) polymerase. Third, J3 has recently been shown to have intermediate and late gene positive transcription elongation factor activity in vivo. Previous experiments have shown that the poly(A) polymerase stimulatory activity and the (nucleoside-2'-O-)methyltransferase activity are two independent functions of the protein that can be genetically separated through site-directed mutagenesis. In this article, the relationship between the J3-mediated transcription elongation activity and the two other functions of the protein was investigated by constructing several site-directed mutant viruses that contain specific defects in either methyltransferase or poly(A) polymerase processivity functions. The results demonstrate that the J3 positive transcription elongation factor activity is a third independent function of the protein that is genetically separable from its two other functions in mRNA modification. The results also show that neither the poly(A) polymerase stimulatory nor the methyltransferase activities of the J3 protein is essential for virus growth in cell culture.
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PMID:The positive transcription elongation factor activity of the vaccinia virus J3 protein is independent from its (nucleoside-2'-O-) methyltransferase and poly(A) polymerase stimulatory functions. 1235 47

Treatment of wild type vaccinia virus infected cells with the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) induces the viral postreplicative transcription apparatus to synthesize longer-than-normal mRNAs through an unknown mechanism. Previous studies have shown that virus mutants resistant to or dependent on IBT affect genes involved in control of viral postreplicative transcription elongation. This study was initiated in order to identify additional viral genes involved in control of vaccinia postreplicative transcription elongation. Eight independent, spontaneous IBT resistant mutants of vaccinia virus were isolated. Marker rescue experiments mapped two mutants to gene G2R, which encodes a previously characterized postreplicative gene positive transcription elongation factor. Three mutants mapped to the largest subunit of the viral RNA polymerase, rpo147, the product of gene J6R. One mutant contained missense mutations in both G2R and A24R (rpo132, the second largest subunit of the RNA polymerase). Two mutants could not be mapped, however sequence analysis demonstrated that neither of these mutants contained mutations in previously identified IBT resistance or dependence genes. Phenotypic and biochemical analysis of the mutants suggests that they possess defects in transcription elongation that compensate for the elongation enhancing effects of IBT. The results implicate the largest subunit of the RNA polymerase (rpo147) in the control of elongation, and suggest that there exist additional gene products which mediate intermediate and late transcription elongation in vaccinia virus.
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PMID:Mapping and phenotypic analysis of spontaneous isatin-beta-thiosemicarbazone resistant mutants of vaccinia virus. 1733 62


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