Characterization of a Second Vaccinia Virus mRNA-Decapping Enzyme Conserved in Poxviruses

The regulation of mRNA turnover allows a cell to rapidly adjust gene expression in an adaptive manner. Vaccinia virus (VACV), the prototype for the laboratory study of poxviruses, modulates both viral and host gene expression during the course of infection (12). The double-stranded DNA genome of VACV encodes approximately 200 proteins that are expressed in sequential stages delineated as early, intermediate, and late (13, 16). Although a transcriptional cascade drives entry into each phase of gene expression, rapid turnover eliminates viral mRNAs soon after their synthesis ceases, thereby augmenting progression through the virus life cycle (1). VACV infection also triggers the degradation of cellular mRNAs (2, 5), which contributes to the shutdown of host protein synthesis. A clue to the mechanism of accelerated cellular and viral mRNA degradation was obtained during studies designed to identify positive transcriptional regulators. Shors and coworkers (18) reported that overexpression of gene VACV-WR_115 (D10R) and to a lesser extent VACV-WR_114 (D9R) decreased expression of viral proteins and the quantity of transcripts possessing a 5′ cap, a structural feature of both cellular and viral messages. In contrast, a mutant virus with a deletion of the D10R gene displays persistence of cellular and viral transcripts, a delay in shutoff of host protein synthesis, and replication defects (14). Taken together, these data suggested that the D10 protein (the product of the D10R gene) regulates mRNA stability and consequently the amount and duration of host and viral proteins synthesized. Although a D9R deletion mutant virus did not exhibit replication defects in tissue culture cells, a mutant virus with deletions of both the D9R and D10R genes could not be isolated, suggesting that these two proteins have compensating functions (14). Moreover, the individual importance of D9 and D10 is reflected in the conservation of the genes for both proteins in most chordopoxviruses. D10 is the more highly conserved of the two, however, as it is one of approximately 50 proteins whose sequence can be recognized in both poxvirus subfamilies: chordopoxviruses and entomopoxviruses. Maintenance of the two genes in chordopoxviruses may be due to different functions or patterns of expression. In this regard, D9 is synthesized early in infection and D10 at late times (8). Thus, continuing synthesis of D9 or D10 may be necessary for efficient replication in vivo. The existence of related functions for D9 and D10 is suggested by their 25% sequence identity to each other and their possession of a Nudix hydrolase or MutT motif, a signature sequence characteristic of a diverse group of phosphohydrolases that cleave nucleoside diphosphates linked to another moiety, X (10). The presence of the Nudix hydrolase motif, in conjunction with the observation that overexpression of D9 and D10 reduced the quantity of capped transcripts, led to the hypothesis that D9 and D10 may cleave the m7GpppN cap structure, marking the message for subsequent degradation by cellular exonucleases. Adding support for this hypothesis, cellular Dcp2 Nudix hydrolases have been shown to modulate mRNA turnover through cleavage of the mRNA cap (19, 20). Recent biochemical investigations demonstrated that purified recombinant VACV D10 has intrinsic mRNA-decapping activity, liberating m7GDP as a product (15). Mutations in the Nudix motif abolished D10 decapping activity, indicating that the site is essential for catalysis and confirming that D10 is the protein responsible for cap cleavage (15). Here we show that D9 also has mRNA-decapping activity, although the two poxvirus Nudix hydrolases appear to have some differences in substrate recognition.