Antibodies directed against an epitope in the N-terminal region of the H4L subunit of the vaccinia virus RNA polymerase inhibit both transcription initiation and transcription termination, in vitro.
The vaccinia virus virion RNA polymerase that is active in early gene transcription contains a unique subunit encoded by the H4L gene. Prior studies demonstrated that this protein is required both for early gene transcription initiation and for transcription termination. Polyclonal antibodies raised against H4L amino acids 1 to 256 prevent both initiation and termination of transcription, in vitro. Pretreatment of the anti-H4L antibody with a H4L fragment containing amino acids 1 to 99 prevents antibody inhibition of both steps, mapping the inhibitory antibody-binding site to this region. A combination of immunoprecipitation and competition studies of antibody binding to wild-type and site-specific mutations of H4L(1-195) mapped the strong epitope to a site that includes Y18. H4L fragments containing an Y18A mutation exhibit diminished ability to block antibody inhibition of transcription initiation and termination. Antibodies inhibit preinitiation complex (PIC) formation but not the activity of preformed PICs, indicating that this region of H4L interacts with one or more factors during active PIC formation. Furthermore, isolated H4L(1-195) directly inhibits PIC activity, supporting this model. Anti-H4L antibody inhibition of transcription termination is only observed in the absence of the essential termination cofactor NPH I. In contrast, antibody inhibition of PIC formation is unaffected by NPH I, demonstrating that the inhibitory antibody and NPH I can bind to H4L at the same time.
Mohamed
Mohamed R
MR
Department of Biochemistry, The Witebsky Center for Microbial Pathogenesis, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, 14214-3000, USA.
Christen
Linda A
LA
Niles
Edward G
EG
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D001667:Binding, Competitive; D002460:Cell Line; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000276:immunology; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D007106:Immune Sera; D009154:Mutation; D013728:Terminator Regions, Genetic / Q000276:immunology; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*
D000914:Antibodies, Viral; D000939:Epitopes; D007106:Immune Sera; D012321:DNA-Directed RNA Polymerases