The viral RNA polymerase H4L subunit is required for Vaccinia virus early gene transcription termination.
Vaccinia virus early gene transcription is catalyzed by a multisubunit virion form of RNA polymerase that possesses a unique subunit, H4L. Prior studies from this laboratory showed that the NH(2)-terminal domain of H4L, containing amino acids 1-195, interacts with the COOH-terminal end of nucleoside triphosphate phosphohydrolase I (NPH I), an ATPase that is employed in early gene transcription termination. Carboxyl-terminal deletion mutations of NPH I lose both the ability to mediate transcription termination and binding to H4L, providing evidence that the interaction between NPH I and H4L is required for termination. In order to test this model further, antibodies raised against segments of H4L were tested for their ability to inhibit transcription termination in vitro. A bead-bound template was employed in these studies, which permitted us to separate transcription initiation from elongation and termination. Antibodies raised against H4L amino acids 1-256 inhibited termination in an in vitro assay using virus-infected cell extracts lacking NPH I, but antibodies raised against H4L amino acids 568-795 did not. Preincubation of anti-H4L(1-256) antibodies with H4L fragments 1-256 or 1-195 prevented antibody inhibition of termination, demonstrating that inhibition was mediated by antibody binding to one or more epitopes in the NH(2)-terminal end of H4L. Antibody inhibition of termination is reduced in wild type virus-infected cell extracts containing NPH I. Furthermore, preincubation of a NPH I minus cell extract with NPH I prior to antibody addition, or readdition of NPH I to isolated ternary complexes prepared in the absence of NPH I, prevented antibody inhibition of transcription termination. These data show that NPH I and the inhibitory antibodies compete for a binding site(s) on H4L, providing further evidence that the H4L subunit of the vaccinia virus RNA polymerase plays a direct role in transcription termination.
Mohamed
M R
MR
Department of Microbiology, Witebsky Center for Microbial Pathogenesis, State University of New York School of Medicine and Biomedical Science, Buffalo, New York 14214, USA.
Niles
E G
EG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002457:Cell Extracts; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic / Q000276:immunology; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*
D000906:Antibodies; D002457:Cell Extracts; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases