VACCINIA VIRUS NUCLEOSIDE TRIPHOSPHATE PHOSPHOHYDROLASE-I CONTROLS EARLY AND LATE GENE-EXPRESSION BY REGULATING THE RATE OF TRANSCRIPTION

We have carried out a detailed analysis of viral mRNAs and proteins pr oduced in cultured cells infected with a temperature-sensitive vaccini a virus mutant (ts36) containing a modified nucleoside triphosphate ph osphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a rec ombinant virus (ts36LUC) which expresses the luciferase marker, we sho wed in seven different cell lines that early expression of the reporte r gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temp erature. The steady-state levels of different early viral polypeptides were also severely reduced. Analysis of steady-state mRNA levels for two early genes (DNA polymerase and D5) showed that inhibition of earl y polypeptide synthesis correlated with a reduction in the levels of m RNA accumulated at the nonpermissive temperature. Analysis of steady-s tate levels of late viral polypeptides and of mRNAs indicated that NPH -I regulation of intermediate and late gene expression is direct and n ot simply a consequence of its role in inhibiting early gene expressio n. Characterization of a rescued virus (R36) demonstrated that the tem perature-sensitive phenotype of ts36 is due solely to the point mutati on in the NPH-I gene. The mutant phenotype is not due to reduced level s of NPH-I present in ts36 virions or to the differential stability of this enzyme in cells infected at the nonpermissive temperature but to inhibition of normal enzymatic activity for this protein. Measurement of viral transcriptional activity in permeabilized purified virions d emonstrated that NPH-I is required for normal rates of transcription i n vaccinia virus. Our findings show ts36 to be a strongly defective ea rly mutant of vaccinia virus and prove that NPH-I plays a key role in the control of early and late virus gene expression, possibly by way o f an auxiliary function which regulates mRNA transcription during the virus growth cycle.