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...