Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression

Abstract

The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (v⌬D10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (v⌬D9). Purified v⌬D10 virions appeared normal by microscopic examination and biochemical analysis but produced 6-to 10-fold-fewer plaques at the same concentration as wild-type or v⌬D9 virions. When 4 PFU per cell of wild-type or v⌬D9 virions or equal numbers of v⌬D10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in v⌬D10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in v⌬D10-infected cells than in cells infected with the wild type or v⌬D9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in v⌬D10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.Vaccinia virus (VACV), the most extensively studied member of the Poxviridae family, contains a large double-stranded DNA genome encoding approximately 200 proteins. Replication occurs in the cytoplasm and is orchestrated in a programmed manner, with sequential transcription of early-, intermediate-, and late-stage genes (25). Numerous positive regulatory factors have been identified, notably the proteins required for stage-specific transcription in conjunction with the viral DNA-dependent RNA polymerase (for a review, see reference 8). The changes in total viral early, intermediate, and late RNA levels, however, suggest the deployment of negative, as well as positive, regulators (3). Indeed, the speed at which RNA levels change can only be explained if viral RNAs have very short half-lives, as was proposed nearly 40 years ago (26, 31). The accelerated turnover of cellular mRNAs during VACV infection (9,19,20,29) might occur by a related or identical degradation mechanism.A putative negative regulator of gene expression was identified in a transfection-based DNA library screen that was successfully used to find activators of VACV late transcription (21, 32). The inhibitory activity was mapped primarily to the D10R gene of VACV, with the D9R gene contributing more modestly to the effect. (Note: VACV genes are designated with a capital letter that describes the HindIII fragment containing the open reading frame, followed by a number that corresp...

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression

“…Previous studies demonstrated that both viral and cellular transcripts decayed more rapidly when D10 was overexpressed (11) and decayed less rapidly when D10 was not expressed (14). However, this finding does not exclude quantitative differences between the decapping of cellular and viral mRNAs.…”

“…The present demonstration of RNA decapping activity by a recombinant VACV D10 fusion protein provides a mechanism to explain in vivo studies that identified VACV D10 as a negative regulator of cellular and viral gene expression (11,14). VACV D10 was predicted to have decapping activity (11) because it contains a Nudix/MutT motif, a sequence characteristic of enzymes that act as nucleotide phosphohydrolases (15).…”

“…Transcript selection might also be influenced by the intracellular localization of the D10. D10 does not localize in viral factory areas (14) where intermediate and late viral transcripts are transcribed and translated (G. Katsafanas and B.M., unpublished data). Transport of D10 from the viral factory could help shield nascent viral transcripts from D10 decapping activity.…”

“…Remarkably, the inhibitory effect of D10 was only manifested on mRNAs that were capped (11), a structural feature of both VACV and host mRNA transcripts (12,13). Deletion of the gene encoding D9 had little effect on virus replication, whereas a D10-deletion mutant virus (v⌬D10) exhibited a 10-fold reduction in the formation of infectious virus (14). When equivalent numbers of virus particles were used for infection, viral and cellular transcripts persisted significantly longer in cells infected with v⌬D10 than with wild-type virus.…”

Investigating Viruses during the Transformation of Molecular Biology

“…Stewart Shuman and Jerry Hurwitz (46) further demonstrated that the transfer of GMP from GTP to the diphosphate end of RNA is a two-step reaction with a VACV enzyme-GMP intermediate. Curiously, VACV also encodes decapping enzymes that regulate mRNA stability (47,48).…”