Article Figures & Data
Figures
- 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 32Pireleased 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 endonucleaseAsp718 (lanes 1 and 2), AvrII (lanes 3, 4, 9, 10, and 11), BstNI (lanes 5, 6, 12, 13, and 14), orEagI (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 theAsp718 site. The P228A and R229A mutations destroy theAvrII 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.
Tables
- Table 1.
Rescue of ts10 by NPH-II alleles containing alanine mutations in motifs II and III
NPH-II gene Virus titer at 40°C (PFU/ml) Sp activity of protein (% of wild-type activity) ATPase Helicase Wild type 1 × 107 100 100 D296A 2 × 104 5 a <5 a E297A 3 × 104 5 a <5 a H299A 3 × 104 58 <1 E300A 1 × 104 1 <1 T326A 1 × 104 51 <1 T328A 1 × 104 52 1 Plasmid <10 ↵a From reference 8.
- Table 2.
Rescue of ts18 by NPH-II alleles containing alanine mutations in motifs I and Ia
NPH-II gene Virus titer at 40°C (PFU/ml) Sp activity of protein (% of wild-type activity) ATPase Helicase Wild type 2 × 107 100 100 T187A 3 × 106 68 52 T192A 4 × 104 <0.2 <1 Q194A 2 × 106 55 46 P228A 8 × 105 91 21 R229A 2 × 104 2 <1 Plasmid <10 - Table 3.
Rescue of ts39 by NPH-II alleles containing alanine mutations in motifs VI
NPH-II gene Virus titer at 40°C (PFU/ml) Sp activity of proteina (% of wild-type activity) ATPase Helicase Wild type 8 × 106 100 100 Q491A 1 × 104 9 2 G494A 1 × 104 10 6 R495A 1 × 104 3 7 G497A 1 × 104 18 10 R498A 1 × 104 3 7 N500A 8 × 106 100 100 P501A 5 × 106 100 100 G502A 6 × 103 18 6 Plasmid <10 ↵a From reference 9.
