Effect of selected mutations in the C-terminal region of the vaccinia virus nucleoside triphosphate phosphohydrolase I on binding to the H4L subunit of the viral RNA polymerase and early gene transcription termination in vitro

Piacente, Sarah C.; Christen, Linda; Mohamed, Mohamed R.; Niles, Edward G.

doi:10.1016/s0042-6822(03)00092-8

Cited by 10 publications

(13 citation statements)

References 27 publications

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“…1A (42). The association of the viral RNA polymerase-associated factor H4 and D11 nucleoside triphosphate phosphohydrolase I (NPHI) was shown to be required for early gene transcription termination, explaining the known restriction of signal-and factor-dependent termination to early genes (107,129). In addition, study of the association between poly(A) polymerase J3, viral RNA polymerase-associated factor H4, and D11 suggests that this interaction may serve as a docking site for J3 in RNA processing (106,107).…”

Section: Virus-virus Protein Interactionsmentioning

confidence: 99%

Poxvirus Proteomics and Virus-Host Protein Interactions

Vliet

Mohamed

Zhang

et al. 2009

Microbiol Mol Biol Rev

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SUMMARY Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.

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Section: Virus-virus Protein Interactionsmentioning

confidence: 99%

Poxvirus Proteomics and Virus-Host Protein Interactions

Vliet

Mohamed

Zhang

et al. 2009

Microbiol Mol Biol Rev

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“…Prior attempts to replace NPH I with A18 for early gene transcription termination were unsuccessful. 3 The intermediate/late form of vaccinia RNA polymerase lacks the early gene-specific subunit Rap94, which interacts with NPH I (19). Instead A18 has been shown to interact with two viral proteins, G2 and H5, involved in transcription regulation of intermediate and late genes (61,62).…”

Section: Discussionmentioning

confidence: 99%

“…The transcription termination assay is similar to the transcript release assay described above except all four NTPs are included in the second step. Under these conditions, termination requires the presence of a U 5 NU signal in the nascent RNA and the viral CE, also known as VTF, in addition to NPH I (1,11,13,19,30).…”

Section: The Ssdna Cofactor Required For Nph I-mediated Transcriptmentioning

confidence: 99%

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Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

Hindman

Gollnick

2016

Journal of Biological Chemistry

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Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I)is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the nontemplate strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the nontemplate strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5 to 3 translocase on single-stranded DNA.

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“…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). The multifunctional J3 protein, with poly(A) polymerase-stimulatory (17), cap nucleoside-2Ј-O-methyltransferase (33), and transcription elongation (23) activities, also interacts with RAP94 (26).…”

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confidence: 99%

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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Effect of selected mutations in the C-terminal region of the vaccinia virus nucleoside triphosphate phosphohydrolase I on binding to the H4L subunit of the viral RNA polymerase and early gene transcription termination in vitro

Cited by 10 publications

References 27 publications

Poxvirus Proteomics and Virus-Host Protein Interactions

Poxvirus Proteomics and Virus-Host Protein Interactions

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

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

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