The nucleoside triphosphatase and helicase activities of vaccinia virus NPH-II are essential for virus replication

Abstract

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.

FIG. 1

Conserved sequence elements define the DExH protein family. The amino acid sequence of motifs I, Ia, II, III, and VI of vaccinia virus NPH-II (GenBank accession no. M35027) is aligned with the sequences (with GenBank accession numbers in parentheses) of the corresponding motifs of six other DExH-box proteins: human helicase A (Hel-A) (L13848); Saccharomyces cerevisiae splicing factors Prp2 (X55936), Prp16 (M31524), and Prp22 (X58681); Drosophila MLE protein (M7412); and HCV RNA helicase NS3 (M62385). Amino acids that have been shown by mutagenesis to be important for the ATPase or helicase activities of NPH-II are shown in shaded boxes. New residues mutated in this study are marked by asterisks.

FIG. 2

NPH-II purification. Aliquots (0.23 μg) of the glycerol gradient preparations of wild-type (WT) NPH-II and the indicated NPH-II-Ala mutants were electrophoresed through an 8% polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized by staining with Coomassie blue dye. The positions and sizes (in kilodaltons) of marker proteins are indicated at the left. The polypeptide corresponding to NPH-II is indicated by the arrow on the right.

FIG. 3

RNA unwinding by wild-type and mutant NPH-II proteins. Helicase assays were performed as described in Materials and Methods. The extent of RNA unwinding by wild-type (WT) and mutated NPH-II proteins is plotted as a function of the amount of input enzyme. Each data point represents the average of two independent determinations. The source of the protein preparation is indicated in the key to symbols.

FIG. 4

Binding of wild-type and mutated His-NPH-II proteins to ssRNA. Binding of NPH-II to a radiolabeled 98-mer ssRNA was measured in a gel shift assay as described in Materials and Methods. The extent of protein-RNA complex formation is plotted as a function of input enzyme. The symbols denoting identities of the proteins used in the titration experiments are as shown in Fig. 3.

FIG. 5

ATP hydrolysis by wild-type (WT) and mutated His-NPH-II proteins. ATP hydrolysis by NPH-II in the presence of single-stranded M13mp18 DNA was assayed as described elsewhere (8). ATPase activity is expressed as nanomoles of ³²P_i released from [γ³²P]ATP during a 30-min incubation at 37°C and is plotted as a function of input enzyme. Each data point represents the average of two independent determinations. The protein preparations used for each titration experiment are indicated in the key to symbols.

FIG. 6

Genotyping of rescued viruses. (A) Summary of viral genotypes determined by restriction analysis of PCR-amplified NPH-II genes from plaque-purified virus revertants. WT, wild type. (B) Genotyping by restriction digestion. Representative restriction endonuclease digests were analyzed by agarose gel electrophoresis. The NPH-II gene was PCR amplified from DNA isolated from cells infected with wild-type vaccinia virus or plaque-purified revertants recovered after transfection with the indicated NPH-II-Ala genes. The 1,718-bp PCR products were digested with restriction endonuclease Asp718 (lanes 1 and 2), AvrII (lanes 3, 4, 9, 10, and 11), BstNI (lanes 5, 6, 12, 13, and 14), or EagI (lanes 15 to 17). For those transfections where the mutant allele was not incorporated into any rescued viruses (R229A, G502A, and T328A), a control restriction digest was performed with the 1,718-bp PCR product amplified from the pTM-based plasmid containing the indicated alanine substitution (lanes 11, 14, and 17). DNA was visualized by staining the agarose gel with ethidium bromide. The positions and sizes (in base pairs) of linear DNA markers are indicated on the left. The 1.7-kbp PCR fragment is depicted as a horizontal bar. The locations of the restriction sites within the wild-type NPH-II gene are denoted below the bar. The Q194A mutation eliminates the Asp718 site. The P228A and R229A mutations destroy the AvrII site. The N500A, P501A, and G502A mutations eliminate the downstream BstNI site. The T328A mutation creates a unique EagI site; digestion at this site (indicated by the arrow above the bar) generates a doublet of 877- and 841-bp fragments.