Abstract
The predicted amino acid sequence of the vaccinia virus gene A18R shows significant homology to the human ERCC3 gene product, which is a member of the DEXH subfamily of the DNA and RNA helicase superfamily II and which plays a role in both RNA polymerase II transcription and nucleotide excision repair of DNA. The vaccinia virus A18R gene product is expressed throughout infection and is encapsidated in virions. Vaccinia virions containing mutant A18R gene product are defective in early viral transcription in vitro, and infection with A18R mutant virus results in aberrant viral transcription late during infection. Thus we hypothesize that the vaccinia virus A18R gene product is a helicase that plays a role in viral transcription and possibly DNA repair. As a first test of this hypothesis, we have affinity purified an amino-terminal polyhistidine-tagged A18R protein and shown that it has DNA-dependent ATPase activity. The A18R ATPase activity is stimulated by both single-stranded and double-stranded DNA and by RNA.DNA hybrids, but not by either single-stranded or double-stranded RNA.
Highlights
Vaccinia virus, the prototype poxvirus, is a large doublestranded! DNA-containing virus that replicates in the cytoplasm of eukaryotic cells [1]
Investigation of the biochemical activity of the vaccinia virus A18R gene product was inspired by computer analysis of the A18R amino acid sequence, which revealed motifs in A18R which are found in the DEXH subfamily of the DNA and RNA helicase superfamily II [22, 23]
In this study we have shown that the vaccinia virus A18R gene product has ATPase activity that is stimulated by either single-stranded DNA (ssDNA), dsDNA, or an RNA·DNA duplex but not by ssRNA or dsRNA
Summary
Vol 270, No., Issue of January 27, pp. 1550-1556, 1995 Printed in U.S.A. (Received for publication, September 15, 1994, and in revised form, November 7, 1994). We hypothesize that the vaccinia virus A18R gene product is a helicase that plays a role in viral transcription and possibly DNA repair. Genetic analysis of vaccinia has identified several additional genes that, when mutated, result in disruption of the normal orderly program of viral gene expression [12,13,14,15,16] Mutants in one such gene, A18R [13], have a complex phenotype, which suggests that the gene plays an important role in transcription throughout vaccinia infection. The homology between A18R and the DEXH family in general or ERCC3 combined with the observations on A18R mutants described above, suggests four alternative hypotheses that describe the role of A18R in vaccinia infection Central to these hypotheses is the notion that A18R is a DNA or RNA helicase or part of a helicase complex. A biochemical analysis ofthe A18R protein should help to clarify its role in vaccinia infection
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