title: Interaction between nucleoside triphosphate phosphohydrolase I and the H4L subunit of the viral RNA polymerase is required for vaccinia virus early gene transcript release.
abstract: Signal-dependent termination is restricted to early poxvirus genes whose transcription is catalyzed by the virion form of RNA polymerase. Two termination factors have been identified. Vaccinia termination factor/capping enzyme is a multifunctional heterodimer that also catalyzes the first three steps of mRNA cap formation and is an essential intermediate gene transcription initiation factor. Nucleoside triphosphate phosphohydrolase I (NPH I) is a single-stranded DNA-dependent ATPase. COOH-terminal deletion mutations of NPH I retain both ATPase and DNA binding activities but are unable either to terminate transcription or to act as dominant negative mutants in vitro. One appealing model posits that the COOH-terminal region of NPH I binds to one or more components in the termination complex. In an attempt to identify NPH I-related protein/protein interactions involved in transcription termination, a series of pull-down experiments were done. Among several vaccinia virus proteins tested, the H4L subunit, unique to the virion form of RNA polymerase, was shown to bind glutathione S-transferase (GST)-NPH I. To further confirm this interaction in virus-infected cells, we constructed recombinant vaccinia virus, vNPHINGST, that expresses GST-tagged NPH I. The H4L subunit of virion RNA polymerase specifically co-purified with GST-NPH I, consistent with a physical interaction. Through the analysis of a series of NH(2)- and COOH-terminal truncation mutations of H4L, the NPH I interaction site was localized to the NH(2)-terminal 195 amino acids of the H4L protein. The H4L binding site on NPH I was mapped to the COOH-terminal region between 457 and 631. Furthermore, COOH-terminal deletion mutations of NPH I failed to bind the NH(2)-terminal region of H4L, explaining their inability to support transcription termination. The COOH-terminal end of NPH I was also shown to be required for transcript release activity and for dominant negative inhibition of release. The requirement for an essential interaction between NPH I and H4L provides an explanation for the observed restriction of transcription termination to early viral genes.
authors.affiliation: Department of Microbiology, the Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York School of Medicine and Biomedical Science at Buffalo, New York 14214, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Mohamed
authors.forename: E G
authors.initials: EG
authors.lastname: Niles
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D043583:Nucleoside-Triphosphatase; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C085819:RAP94 protein, Vaccinia virus; D012333:RNA, Messenger; D014157:Transcription Factors; D014764:Viral Proteins; C064449:early transcription factor protein, Vaccinia virus; D005982:Glutathione Transferase; D012321:DNA-Directed RNA Polymerases; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase