RNA polymerase-associated transcription specificity factor encoded by vaccinia virus.

Ahn, Bokyung; Moss, Bernard

doi:10.1073/pnas.89.8.3536

Cited by 87 publications

(50 citation statements)

References 34 publications

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“…Based on the above data, A19 could interact directly with RAP94 or RNA polymerase, since the two proteins are tightly associated (30,38). To resolve this issue, the gene encoding FS-A19 was inserted into the genome of the RAP94 (H4)-inducible virus vRAP94i.…”

Section: Resultsmentioning

confidence: 99%

Interactions of the Vaccinia Virus A19 Protein

Satheshkumar¹,

Olano²,

Hammer³

et al. 2013

J Virol

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The A19 protein of vaccinia virus (VACV) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. Multiple-sequence alignments of A19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of CXXC motifs that suggested a zinc finger or redox active sites. The importance of the CXXC motif was confirmed by cysteine-toserine substitutions, which rendered the altered protein unable to trans-complement infectivity of a null mutant. Nevertheless, the cysteines were not required for function of the poxvirus-specific redox pathway. Epitope-tagged A19 proteins were detected in the nucleus and cytoplasm in both infected and uninfected cells, but this distribution was unaffected by alanine substitutions of the arginine residues, which only partially reduced the ability of the mutated protein to trans-complement infectivity. Viral proteins specifically associated with affinity-purified A19 were identified by mass spectrometry as components of the transcription complex, including RNA polymerase subunits, RAP94 (RNA polymerase-associated protein 94), early transcription factors, capping enzyme, and nucleoside triphosphate phosphohydrolase I, and two core proteins required for morphogenesis. Further studies suggested that the interaction of A19 with the RNA polymerase did not require RAP94 or other intermediate or late viral proteins but was reduced by mutation of cysteines in the putative zinc finger domain. Although A19 was not required for incorporation of the transcription complex in virus particles, the transcriptional activity of A19-deficient virus particles was severely reduced.

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Section: Resultsmentioning

confidence: 99%

Interactions of the Vaccinia Virus A19 Protein

Satheshkumar¹,

Olano²,

Hammer³

et al. 2013

J Virol

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“…Although RAP94 behaves as an RPO subunit, it differs from the other subunits in being expressed at late times after infection, consistent with its exclusive role in early transcription. The RAP94 requirement for early transcription led to a proposal that it forms a bridge between the core RPO and the early transcription factor VETF (3,14). Further studies showed that RAP94 is present in the ternary complex (2,14) and remains associated with RPO during elongation (14).…”

Section: Resultsmentioning

confidence: 99%

“…An alternate possibility is that VETF can interact directly with NPH I as well as RAP94, although the former has not been demonstrated. RPO, with the addition of the RAP94 polypeptide, is used for early gene expression (3,21). Although RAP94 behaves as an RPO subunit, it differs from the other subunits in being expressed at late times after infection, consistent with its exclusive role in early transcription.…”

Section: Resultsmentioning

confidence: 99%

“…Rabbit polyclonal antisera for D6, A7, RAP94, and RPO30 were described previously (1,3,18). Rabbit polyclonal antiserum for NPH I was obtained from Edward Niles (SUNY, Buffalo, NY).…”

Section: Cells and Viruses Bs-c-1 Cells (Atccmentioning

confidence: 99%

“…The finding that VETF is packaged under the latter conditions, possibly by binding to promoter sequences, suggested a model whereby RAP94 links VETF with RPO and other enzymes. RAP94 is tightly associated with RPO and is required for early transcription by contributing to promoter specificity (2,3,14,21) as well as for transcription termination (28). NPH I, a DNA-dependent ATPase (31) required for the termination of early transcripts (12,13), associates directly with RAP94 (27,32).…”

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confidence: 99%

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Interaction of the Vaccinia Virus RNA Polymerase-Associated 94-Kilodalton Protein with the Early Transcription Factor

Yang

Moss

2009

J Virol

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A multisubunit RNA polymerase (RPO) encoded by vaccinia virus (VACV), in conjunction with specific factors, transcribes early, intermediate, and late viral genes. However, an additional virus-encoded polypeptide referred to as the RPO-associated protein of 94 kDa (RAP94) is tightly bound to the RPO for the transcription of early genes. Unlike the eight RPO core subunits, RAP94 is synthesized exclusively at late times after infection. Furthermore, RAP94 is necessary for the packaging of RPO and other components needed for early transcription in assembling virus particles. The direct association of RAP94 with NPH I, a DNA-dependent ATPase required for transcription termination, and the multifunctional poly(A) polymerase small subunit/2-O-methyltransferase/elongation factor was previously demonstrated. That RAP94 provides a structural and functional link between the core RPO and the VACV early transcription factor (VETF) has been suspected but not previously demonstrated. Using VACV recombinants that constitutively or inducibly express VETF subunits and RAP94 with affinity tags, we showed that (i) VETF associates only with RPO containing RAP94 in vivo and in vitro, (ii) the association of RAP94 with VETF requires both subunits of the latter, (iii) neither viral DNA nor other virus-encoded late proteins are required for the interaction of RAP94 with VETF and core RPO subunits, (iv) different domains of RAP94 bind VETF and core subunits of RPO, and (v) NPH I and VETF bind independently and possibly simultaneously to the N-terminal region of RAP94. Thus, RAP94 provides the bridge between the RPO and proteins needed for transcription initiation, elongation, and termination.The ability of poxviruses to replicate in the cytoplasm depends on the presence within the infectious particle of a system for the synthesis of translatable early mRNAs (29). The protein components of this system have been isolated from vaccinia virus (VACV) and comprise a multisubunit DNA-dependent RNA polymerase (RPO), RPO-associated protein of 94 kDa (RAP94), the VACV early transcription factor (VETF), capping and methylating enzymes, poly(A) polymerase, two nucleic acid-dependent ATPases, and topoisomerase. The products of the early mRNAs include proteins needed for the replication of the viral genome and intermediate gene transcription factors, thereby allowing the next stage of gene expression. Newly synthesized viral DNA serves as a template for the successive transcription of intermediate genes encoding late transcription factors and late genes encoding early transcription factors. In this way, the orderly expression of early, intermediate, and late genes is tightly programmed.Previous studies suggested that the components of the early transcription system are associated in a labile complex. The complex, which could initiate and terminate transcription from an exogenous DNA template containing an early promoter, was released by the disruption of purified virions and isolated by glycerol gradient sedimentation (8). After further treatments and...

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Apoptosis Induced by a Postbinding Step of Vaccinia Virus Entry into Chinese Hamster Ovary Cells

Ramsey-Ewing

Moss

1998

Virology

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Unlike most cell types examined, nonpermissive Chinese hamster ovary (CHO) cells readily underwent apoptosis upon infection with vaccinia virus (VV). Apoptosis was observed as early as 3 h postinfection with an electrophoretic assay of DNA fragmentation and by 8 h using an in situ (TUNEL) assay. The CHO hr gene from cowpox virus, which overcomes host range restriction of VV in CHO cells, merely delayed the onset of apoptosis by approximately 3 h. Intermediate and late viral protein synthesis were not necessary for apoptosis since these events do not proceed under nonpermissive conditions. Apoptosis also occurred in the presence of cytosine arabinoside or cycloheximide, which inhibits DNA or protein synthesis, respectively, and after infection with a mutant virus that is blocked in early transcription. We also demonstrated that viral early transcription was not required for induction of apoptosis by infecting CHO cells with psoralen/UV-inactivated virus. On the other hand, apoptosis was inhibited by a neutralizing antibody to the virion L1R protein added either before or after virus attachment to cells. These results indicate that a postbinding step associated with cell entry is sufficient and required for induction of apoptosis in CHO cells. Recent progress on apoptotic signaling pathways raises the possibility that a cellular receptor for VV may be involved in apoptosis.

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RNA polymerase-associated transcription specificity factor encoded by vaccinia virus.

Cited by 87 publications

References 34 publications

Interactions of the Vaccinia Virus A19 Protein

Interactions of the Vaccinia Virus A19 Protein

Interaction of the Vaccinia Virus RNA Polymerase-Associated 94-Kilodalton Protein with the Early Transcription Factor

Apoptosis Induced by a Postbinding Step of Vaccinia Virus Entry into Chinese Hamster Ovary Cells

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