Vaccinia virus nucleoside triphosphate phosphohydrolase I controls early and late gene expression by regulating the rate of transcription

Abstract

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.

Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene

“…Unlike K3L, D11L and A18R are essential genes for VV replication in tissue culture. These two genes, encode functions which may indirectly affect sensitivity to IFN by their involvement in virus-specific transcriptional control (2,9). This level of control would therefore maintain lower levels of dsRNA in infected cells.…”

The dual-specificity phosphatase encoded by vaccinia virus, VH1, is essential for viral transcription in vivo and in vitro

“…The known components of the early transcription machinery include a heterodimeric transcription factor (vETF), a seven-subunit RNA polymerase, the RAP94 polymerase accessory protein, the heterodimeric capping enzyme/ termination protein, and the heterodimeric poly(A) polymerase, one of whose subunits also functions as a methyltransferase during mRNA cap maturation (32). An encapsidated RNA helicase is also essential for transcription (45), and two other encapsidated nucleoside triphosphatases are reported to affect transcription in vivo and/or in vitro (5,15,24,47). None of the proteins involved in transcription have been reported to be phosphorylated in vivo.…”

Interaction of the Vaccinia Virus Nucleoside Triphosphate Phosphohydrolase I with Linear Oligonucleotides

“…Phenotypic characterization of the ts mutations indicates that at the nonpermissive temperature virus intermediate and late gene transcription is impaired (). Notable decreases in the levels of both early gene transcription and DNA replication can be observed in some cell lines ( , ). These pleiotropic effects observed in the NPH I ts mutations may indicate that NPH I serves multiple functions in the viral life cycle.…”