MUTATIONAL ANALYSIS OF VACCINIA VIRUS NUCLEOSIDE TRIPHOSPHATE PHOSPHOHYDROLASE-II, A DEXH BOX RNA HELICASE

Vaccinia virus nucleoside triphosphate phosphohydrolase II (NPH-II), a 3'-to-5' RNA helicase, displays sequence similarity to members of the DExH family of nucleic acid dependent nucleoside triphosphatases (NTP ases). The contributions of the conserved GxGKT and DExH motifs to enz yme activity were assessed by alanine scanning mutagenesis. Histidine- tagged versions of NPH-II were expressed in vaccinia virus-infected BS C40 cells and purified by nickel affinity and conventional fractionati on steps. Wild-type His-NPH-II was indistinguishable from native NPH-I I with respect to RNA helicase, RNA binding, and nucleic acid-stimulat ed NTPase activities. The K-191-->A (K191A), D296A, and E297A mutant p roteins bound RNA as well as wild-type His-NPH-II did, but they were s everely defective in NTPase and helicase functions. The H299A mutant w as active in RNA binding and NTP hydrolysis but was defective in duple x unwinding. Whereas the NTPase of wild-type NPH-II. was stimulated >1 0-fold by polynucleotide cofactors, the NTPase of the H299A mutant was nucleic acid independent. Because the specific NTPase activity of the H299A mutant in the absence of nucleic acid was near that of wild-typ e enzyme in the presence of DNA or RNA and because the K-m for ATP was unaltered by the H299A substitution, we regard this mutation as a ''g ain-of-function'' mutation and suggest that the histidine residue in t he DExH box is required to couple the NTPase and helicase activities.