Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription.
Temperature-sensitive mutations (ts10, ts18, and ts39) of the vaccinia virus RNA helicase nucleoside triphosphate phosphohydrolase II (NPH-II) result in the production of noninfectious progeny virions at the restrictive temperature. The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of Western blot (immunoblot) analysis indicate that these particles lack NPH-II, whereas other enzymatic components of the virus core are present. These components include the following: DNA-dependent RNA polymerase subunits rpo147, rpo132, rpo94, rpo35, rpo30, rpo22, and rpo18; early transcription initiation factor subunits A8 and D6; mRNA capping enzyme subunits D1 and D12; RNA cap 2'-O-methyltransferase; A18 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase. Although RNA polymerase is encapsidated by the mutant viruses, mRNA synthesis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporation into acid-insoluble material. Moreover, the transcripts synthesized by the mutant particles are longer than normal and remain virion associated. Transcription initiation by mutant virions occurs accurately at an endogenous genomic promoter, albeit at reduced levels (1 to 7%) compared with that of wild-type virions. In contrast, extracts of the mutant virions catalyze the wild-type level of transcription from an exogenous template containing an early promoter. We conclude that NPH-II is required for early mRNA synthesis uniquely in the context of the virus particle. Possible roles in transcription termination and RNA transport are discussed.
Gross
C H
CH
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
S
S
D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D010455:Peptides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D011817:Rabbits; D013696:Temperature; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism; D014771:Virion
D010455:Peptides; D012367:RNA, Viral; D014764:Viral Proteins; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases