Temperature-sensitive mutations in the vaccinia virus H4 gene encoding a component of the virion RNA polymerase

Kane, Eileen; Shuman, Stewart

doi:10.1128/jvi.66.10.5752-5762.1992

Cited by 42 publications

(20 citation statements)

References 45 publications

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“…In addition to synthesis of viral late proteins, morphologically mature virions were assembled in cells infected with mutant virus in the absence of inducer. This phenotype was similar to that caused by a temperature sensitivity mutation in the H4 gene (25). To obtain sufficient amounts of our mutant virions for characterization, we scaled up their production in HeLa cell suspension cultures.…”

Section: Discussionmentioning

confidence: 81%

“…Open reading frame (ORF) H4, encoding RAP94, is expressed late in infection and has a short region similar to that of eukaryotic RNA polymerase-associated general transcription factor RAP30 (2).. RAP94 is required for transcription of early genes in vitro, but on the basis of its time of synthesis in infected cells, is unlikely to be involved in either intermediate or late gene expression (2). Several temperaturesensitive viruses with mutations in H4 express viral gene products, and at least one produces normal-appearing virions under nonpermissive conditions (9,25). Here, we describe the construction and characterization of a vaccinia virus mutant in which the H4 promoter was down regulated by a nucleotide substitution and conditionally repressed by tandem copies of the Escherichia coli lac operator.…”

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confidence: 99%

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Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein

Zhang

Ahn

Moss

1994

J Virol

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When expression of the vaccinia virus gene encoding RAP94 (a protein that is associated with the viral multisubunit RNA polymerase and confers transcriptional specificity for early promoters) was repressed, the infectious virus yield was reduced by more than 99%. Nevertheless, intermediateand late-stage viral gene expression and formation of ultrastructurally mature, membrane-enveloped virions occurred under the nonpermissive conditions. The RAP94-deficient particles contained the viral genome, structural proteins, early transcription factor, and certain enzymes but, unlike normal virions, had low or undetectable amounts of the viral RNA polymerase, capping enzyme/termination factor, poly(A) polymerase, DNA-dependent ATPase, RNA helicase, and topoisomerase. The presence of these viral enzymes in the cytoplasm indicated that RAP94 is required for targeting a complex of functionally related proteins involved in the biosynthesis of mRNA.

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Section: Discussionmentioning

confidence: 81%

mentioning

confidence: 99%

Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein

Zhang

Ahn

Moss

1994

J Virol

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“…Chemical modification of the base, the 2' position of the sugar or the phosphodiester bond impedes termination, as well, demonstrating the remarkable specificity for a U-rich sequence (Shuman and Moss, 1988;1989;Deng and Shuman, 1997a;Mohamed et al, 2006). UV photo crosslinking studies demonstrated that the UUUUUNU motif is capable of interacting with both Rap94 (Christen et al, 2008), an early gene specific RNA polymerase subunit that is required for both early gene transcription initiation (Ahn et al, 1994;Kane and Shuman, 1992;Ahn and Moss, 1992;Deng and Shuman, 1994) and termination Niles, 2000, 2001;Mohamed et al, 2002), and with VTF, the vaccinia virus early gene transcription termination factor (Hagler et al, 1994;Christen et al, 2008). VTF is a heterodimeric enzyme that also catalyzes the initial three steps in mRNA cap formation (Ensinger et al, 1975;Venkatesan et al, 1980) and serves as an essential intermediate gene transcription initiation factor (Vos et al, 1991).…”

Section: )mentioning

confidence: 99%

Determinants of vaccinia virus early gene transcription termination

Piacente¹,

Christen²,

Dickerman³

et al. 2008

Virology

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Vaccinia virus early gene transcription requires the vaccinia termination factor, VTF, nucleoside triphosphate phosphohydrolase I, NPH I, ATP, the virion RNA polymerase, and the motif, UUUUUNU, in the nascent RNA, found within 30 to 50 bases from the poly A addition site, in vivo. In this study, the relationships among the vaccinia early gene transcription termination efficiency, termination motif specificity, and the elongation rate were investigated. A low transcription elongation rate maximizes termination efficiency and minimizes specificity for the UUUUUNU motif. Positioning the termination motif over a 63 base area upstream from the RNA polymerase allowed efficient transcript release, demonstrating a remarkable plasticity in the transcription termination complex. Efficient transcript release was observed during ongoing transcription, independent of VTF or UUUUUNU, but requiring both NPH I and either ATP or dATP. This argues for a two step model: the specifying step, requiring both VTF and UUUUUNU, and the energy-dependent step employing NPH I and ATP. Evaluation of NPH I mutants for the ability to stimulate transcription elongation demonstrated that ATPase activity and a stable interaction between NPH I and the Rap94 subunit of the viral RNA polymerase are required. These observations demonstrate that NPH I is a component of the elongating RNA polymerase, which is catalytically active during transcription elongation.

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“…Early genes are transcribed within the infecting virus core utilizing the core-associated RNA polymerase and transcription factors. The core actually contains two multi-subunit RNA polymerases (Ahn and Moss, 1992;Kane and Shuman, 1992). One form possesses eight virus encoded subunits and exhibits the general architecture of an eukaryotic nuclear RNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

Vaccinia virus early gene transcription termination factors VTF and Rap94 interact with the U9 termination motif in the nascent RNA in a transcription ternary complex

et al. 2008

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The vaccinia virus core contains a 195 kb double stranded DNA genome, a multi-subunit RNA polymerase, transcription initiation and termination factors and mRNA processing enzymes. Upon infection, vaccinia virus early gene transcription takes place in the virus core. Transcription initiates at early promoters and terminates in response to a termination motif, UUUUUNU, in the nascent mRNA. Early gene transcription termination requires the vaccinia virus termination factor, VTF, a single stranded DNA-dependent ATPase, and NPH I, the Rap94 subunit of the virion RNA polymerase, as well as the presence of the UUUUUNU motif in the nascent RNA. The position of UUUUUNU in the ternary complex suggests that it serves as a site of interaction with one or more components of the transcription termination complex. In order to identify the factor(s) that interact with UUUUUNU a series of direct UV photo crosslinking and ribonuclease A protection studies were undertaken. Through these analyses both VTF and Rap94 were shown to interact with UUUUUNU in the isolated ternary complex. Evidence indicates that the interaction is not mutually exclusive. VTF was shown to bind to UUUUUNU through the N-terminal domain of the large D1 subunit. Furthermore, VTF protects from RNAse A digestion both the 5' region of the nascent transcript as well as a large central component containing UUUUUNU. The addition of an oligonucleotide containing the (5Br)U9 sequence both directly inhibits transcription termination, in vitro and inhibits UV photo crosslinking of VTF to the nascent RNA in the ternary complex. These results support a model in which the availability of the UUUUUNU motif outside of the transcribing RNA polymerase permits binding of both transcription termination factors, VTF and Rap94, to UUUUUNU. The assembly of this termination complex initiates the transcription termination sequence.

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Temperature-sensitive mutations in the vaccinia virus H4 gene encoding a component of the virion RNA polymerase

Cited by 42 publications

References 45 publications

Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein

Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein

Determinants of vaccinia virus early gene transcription termination

Vaccinia virus early gene transcription termination factors VTF and Rap94 interact with the U9 termination motif in the nascent RNA in a transcription ternary complex

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