The nucleoside triphosphatase and helicase activities of vaccinia virus NPH-II are essential for virus replication.
Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by six shared sequence motifs. The contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia (PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by alanine scanning. NPH-II-Ala proteins were expressed in baculovirus-infected Sf9 cells, purified, and characterized with respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. In contrast, alanine mutations at His-299 (motif II) and at Thr-326 and Thr-328 (motif III) elicited defects in RNA unwinding but spared the ATPase. None of the mutations analyzed affected the binding of NPH-II to RNA. These findings, together with previous mutational studies, indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif III and the His moiety of the DExH-box serve to couple the NTPase and helicase activities. Wild-type and mutant NPH-II-Ala genes were tested for the ability to rescue temperature-sensitive nph2-ts viruses. NPH-II mutations that inactivated the phosphohydrolase in vitro were lethal in vivo, as judged by the failure to recover rescued viruses containing the Ala substitution. The NTPase activity was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which NTPase was uncoupled from unwinding (H299A, T326A, and T328A) were also lethal. We conclude that the phosphohydrolase and helicase activities of NPH-II are essential for virus replication.
Gross
C H
CH
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
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S
D017766:Acid Anhydride Hydrolases / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D008969:Molecular Sequence Data; D009154:Mutation; D043583:Nucleoside-Triphosphatase; D011485:Protein Binding / Q000235:genetics; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000502:physiology*; D012367:RNA, Viral / Q000378:metabolism; D014616:Vaccinia virus / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology*; D016335:Zinc Fingers
D012367:RNA, Viral; D012316:RNA Nucleotidyltransferases; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases