The D10 Decapping Enzyme of Vaccinia Virus Contributes to Decay of Cellular and Viral mRNAs and to Virulence in Mice

Abstract

bPosttranscriptional mechanisms are important for regulation of cellular and viral gene expression. The presence of the 5= cap structure m 7 G(5=)ppp(5=)Nm is a general feature of mRNAs that provides protection from exoribonuclease digestion and enhances translation. Vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. Decapping is mediated by two related enzymes, D9 and D10, which are synthesized before and after viral DNA replication, respectively. The timing of D10 synthesis correlates better with the shutdown of host gene expression, and deletion of this gene has been shown to cause persistence of host and viral mRNAs in infected cells. Here, we constructed specific mutant viruses in which translation of D10 was prevented by stop codons or activity of D10 was abrogated by catalytic site mutations, without other genomic alterations. Both mutants formed plaques of normal size and replicated to similar extents as the parental virus in monkey epithelial cells and mouse embryonic fibroblasts. The synthesis of viral proteins was slightly delayed, and cellular and viral mRNAs persisted longer in cells infected with the mutants compared to either the parental virus or clonal revertant. Despite the mild effects in vitro, both mutants were more attenuated than the revertants in intranasal and intraperitoneal mouse models, and less infectious virus was recovered from organs. In addition, there was less lung histopathology following intranasal infection with mutant viruses. These data suggest that the D10 decapping enzyme may help restrict antiviral responses by accelerating host mRNA degradation during poxvirus infection. P osttranscriptional mechanisms are important for the regulation of cellular and viral gene expression at the levels of RNA stability and translation. The presence of a 5= cap structure m 7 G(5=)ppp(5=)Nm is a general feature of eukaryotic mRNAs and many viral mRNAs that provides protection from exonuclease digestion and enhances translation (1-6). In eukaryotic cells, mRNA decay begins with shortening of the poly(A) tail and proceeds in either the 5=-to-3= or 3=-to-5= direction. The latter is mediated by the cytoplasmic RNA exosome (7-9) and a scavenger enzyme that degrades the cap (8). In the 5=-to-3= pathway, removal of the cap (10-13) is followed by exoribonuclease Xrn1 digestion (14,15). Enzymes with nudix hydrolase motifs that decap cytoplasmic mRNA are present in Saccharomyces cerevisiae and mammalian cells and are thought to function in mRNA decay (10-13, 16, 17).Degradation of cellular mRNA may be advantageous to viruses by decreasing competition for the translational machinery and by reducing the synthesis of factors that contribute to the innate and adaptive immune responses to infection. The ability of viruses to accelerate mRNA decay has been intensively studied with members of the herpesvirus family, which replicate in the nucleus and utilize the transcriptional machinery of the cell (18, 19). For alphaherpesviruses, accelerated mRNA turnover is mediat...

Myxoma Virus Induces Ligand Independent Extrinsic Apoptosis in Human Myeloma Cells

“…In MYXV, host-protein shutoff is mediated by the two poxviral decapping enzymes, M084 and M085 57 . These enzymes remove the 5’ cap from viral and cellular mRNA’s thus preventing their translation 58, 59 . We have recently characterized a MYXV mutant (vMYX-ΔM083) which has enhanced expression of M084 and M085 due to the local insertion of a strong poxviral promoter 25 .…”

Redundancy complicates the definition of essential genes for vaccinia virus

“…However, five conserved genes (G6R, I5L, F16L, D10R and A14.5L) have been defined as non-essential because single-gene-deletion mutants were created successfully (Betakova et al, 2000;Parrish & Moss, 2006;Senkevich et al, 2008;Sood et al, 2008;Senkevich et al, 2011;Liu et al, 2014). Four of these genes are known to affect virulence after infection of mice, suggesting that these genes were conserved because they carry out a crucial role in vivo (Betakova et al, 2000;Senkevich et al, 2008;Sood et al, 2008;Liu et al, 2014 (Meng et al, 2009). Other functions such as inhibition of apoptosis are redundant to some degree as well, with a hierarchy of importance across viral genes .…”

Comparing the functions of equine and canine influenza H3N8 virus PA-X proteins: Suppression of reporter gene expression and modulation of global host gene expression

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