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-I I) result in the production of noninfectious progeny virions at the re strictive temperature, The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judg ed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The r esults of Western blot (immunoblot) analysis indicate that these parti cles lack NPH-II, whereas other enzymatic components of the virus core are present, These components include the following: DNA-dependent RN A 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; A1 8 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase, Alt hough RNA polymerase is encapsidated by the mutant viruses, mRNA synth esis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporat ion into acid-insoluble material, Moreover, the transcripts synthesize d by the mutant particles are longer than normal and remain virion ass ociated, 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 e xogenous template containing an early promoter, We conclude that NPH-I I is required for early mRNA synthesis uniquely in the context of the virus particle, Possible roles in transcription termination and RNA tr ansport are discussed.