Characterization of a second vaccinia virus mRNA-decapping enzyme conserved in poxviruses.
Vaccinia virus (VACV) encodes enzymes that cap the 5' end of viral mRNAs, which enhances their stability and translation. Nevertheless, recent studies demonstrated that the VACV D10 protein (VACV-WR_115) decaps mRNA, an enzymatic activity not previously shown to be encoded by a virus. The decapping activity of D10 is dependent on a Nudix hydrolase motif that is also present in the VACV D9 protein (VACV-WR_114), which shares 25% sequence identity with D10. Here, we showed that a purified recombinant VACV D9 fusion protein also decaps mRNA and that this activity was abolished by point mutations in the Nudix hydrolase motif. Decapping was specific for a methylated cap attached to RNA and resulted in the liberation of m7GDP. D9 differed from D10 in requiring a longer capped RNA substrate for optimal activity, having greater sensitivity to inhibition by uncapped RNA, and having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA. Since D9 is expressed early in infection and D10 late, we suggest that the two proteins enhance mRNA turnover and manipulate gene expression in a complementary and overlapping manner.
Parrish
Susan
S
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 33 North Drive, Bethesda, MD 20892-3210, USA.
Moss
Bernard
B
D020816:Amino Acid Motifs / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017354:Point Mutation; D012315:RNA Caps / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D012315:RNA Caps; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C030052:mRNA decapping enzymes; C111106:7-methylguanosine 5'-diphosphate; D006153:Guanosine Diphosphate; D004722:Endoribonucleases