SUMMARY Poxvirus replication involves synthesis of double stranded RNA (dsRNA), which can trigger antiviral responses by inducing phosphorylation-mediated activation of protein kinase R (PKR) and stimulating 2’5’-oligoadenylate synthetase (OAS). PKR inactivates the translation initiation factor eIF2α via phosphorylation, while OAS induces the endonuclease RNase L to degrade RNA. We show that poxvirus decapping enzymes D9 and D10, which remove caps from mRNAs, inhibit these antiviral responses by preventing dsRNA accumulation. Catalytic site mutations of D9 and D10, but not of either enzyme alone, halt vaccinia virus late protein synthesis and inhibit virus replication. Infection with the D9-D10 mutant was accompanied by massive mRNA reduction, cleavage of ribosomal RNA and phosphorylation of PKR and eIF2α that correlated with a ~15-fold increase in dsRNA compared to wild-type virus. Additionally, mouse studies show extreme attenuation of the mutant virus. Thus, vaccinia virus decapping, in addition to targeting mRNAs for degradation, prevents dsRNA accumulation and anti-viral responses.

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...

The African Swine Fever Virus (ASFV) encodes a single Nudix enzyme in its genome, termed the g5R protein (g5Rp). Nudix phosphohydrolases cleave a variety of substrates, such as nucleotides and diphosphoinositol polyphosphates. Previously, ASFV g5Rp was shown to hydrolyze diphosphoinositol polyphosphates and GTP, but was unable to cleave methylated mRNA cap analogues. In vaccinia virus (VACV), a distant relative of ASFV, the D9 and D10 Nudix enzymes were shown to cleave the mRNA cap, but only when the cap was attached to an RNA body. Here, we show that recombinant ASFV g5Rp hydrolyzes the mRNA cap when tethered to an RNA moiety, liberating m7GDP as product. Mutations in the Nudix motif abolished mRNA decapping activity, confirming that g5Rp was responsible for cap cleavage. The decapping activity of g5Rp was potently inhibited by excess uncapped RNA but not by methylated cap analogues, suggesting that substrate recognition occurs by RNA binding.

Two well known fuzzy partition clustering algorithms, FCM and FPCM are based on Euclidean distance function, which can only be used to detect spherical structural clusters. GK clustering algorithm and GG clustering algorithm, were developed to detect non-spherical structural clusters, but both of them need additional prior information. In our previous studies, we developed four improved algorithms, FCM-M, FPCM-M, FCM-CM and FPCM-CM based on unsupervised Mahalanobis distance without any additional prior information. In first two algorithms, only the local covariance matrix of each cluster was considered, In last two algorithms, not only the local covariance matrix of each cluster but also the overall covariance matrix was considered, and FPCM-CM is the better one. In this paper, a more information about "separable criterion" is considered, and the further improved new algorithm, "fuzzy possibility c-mean based on complete Mahalanobis distance and separable criterion, (FPCM-CMS)" is proposed. It can get more information and higher accuracy by considering the additional separable criterion than FPCM-CM. A real data set was applied to prove that the performance of the FPCM-CMS algorithm is better than those of above six algorithms.