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 produced in cultured cells infected with a temperature-sensitive vaccinia virus mutant (ts36) containing a modified nucleoside triphosphate phosphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a recombinant virus (ts36LUC) which expresses the luciferase marker, we showed in seven different cell lines that early expression of the receptor gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temperature. 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 early polypeptide synthesis correlated with a reduction in the levels of mRNA accumulated at the nonpermissive temperature. Analysis of steady-state levels of late viral polypeptides and of mRNAs indicated that NPH-I regulation of intermediate and late gene expression is direct and not simply a consequence of its role in inhibiting early gene expression. Characterization of a rescued virus (R36) demonstrated that the temperature-sensitive phenotype of ts36 is due solely to the point mutation in the NPH-I gene. The mutant phenotype is not due to reduced levels 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 demonstrated that NPH-I is required for normal rates of transcription in vaccinia virus. Our findings show ts36 to be a strongly defective early 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 of an auxiliary function which regulates mRNA transcription during the virus growth cycle.
Diaz-Guerra
M
M
Department of Biochemistry, State University of New York, Health Science Center at Brooklyn 11203.
Esteban
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D017930:Genes, Reporter; D006358:Hot Temperature; D006801:Humans; D008156:Luciferases; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011755:Pyrophosphatases / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014771:Virion / Q000378:metabolism
D012333:RNA, Messenger; D008156:Luciferases; D011755:Pyrophosphatases; C021895:nucleoside triphosphate pyrophosphatase