Abstract
UL9, an essential gene for herpes simplex virus type 1 (HSV-1) DNA replication, exhibits helicase and origin DNA binding activities. It has been hypothesized that UL9 binds and unwinds the HSV-1 origin of replication, creating a replication bubble and promoting the assembly of the viral replication machinery; however, direct confirmation of this hypothesis has not been possible. Based on the presence of conserved helicase motifs, UL9 has been classified as a superfamily II helicase. Mutations in conserved residues of the helicase motifs I-VI of UL9 have been isolated, and most of them fail to complement a UL9 null virus in vivo (Martinez R., Shao L., and Weller S. (1992) J. Virol. 66, 6735-6746). In addition, mutants in motifs I, II, and VI were found to be transdominant (Malik, A. K., and Weller, S. K. (1996) J. Virol. 70, 7859-7866). Here we present the characterization of the biochemical properties of the UL9 helicase motif mutants. We report that mutations in motifs I-IV and VI affect the ATPase activity, and all but the motif III mutation completely abolish the helicase activity. In addition, mutations in these motifs do not interfere with UL9 dimerization or the ability of UL9 to bind the HSV-1 origin of replication. Based on the similarity of the helicase motif sequences between UL9 and UvrB, another superfamily II member with helicase-like activity, we were able to map the UL9 mutations on the structure of the UvrB protein and provide an explanation for the observed phenotypes. Our results indicate that the helicase function of UL9 is indispensable for viral replication, supporting the hypothesis that UL9 is essential for unwinding the HSV-1 origin of replication in vivo. Furthermore, the data presented provide insights into the mechanism of transdominance of the UL9 helicase motif mutants.
Highlights
The mechanism of helicase action is believed to involve the coupling between several steps including ATP binding, ATP hydrolysis, and binding to nucleic acid
We report that mutations in motifs I–IV and VI affect the ATPase activity, and all but the motif III mutation completely abolish the helicase activity
Our results indicate that the helicase function of UL9 is indispensable for viral replication, supporting the hypothesis that UL9 is essential for unwinding the herpes simplex virus type 1 (HSV-1) origin of replication in vivo
Summary
Reagents and Materials—Supplemented Grace’s medium and penicillin/streptomycin were purchased from Life Technologies, Inc.; fetal calf serum was from Gemini Biological Products. Limited Proteolysis—Protease inhibitors used during the protein purification scheme were removed by chromatography on a Sephadex G-75 gel filtration column using 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT buffer and flow rate of 1 ml/min. Each reaction contained 1ϫ filter binding buffer (50 mM HEPES, pH 7.6, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10% glycerol), 0.2 mM NaCl, 0.4 nM DNA substrate (unless otherwise stated), 100ϫ (w/w) excess of poly(dI-dC), and 5 mg/ml BSA. Nitrocellulose membranes were incubated for 2 h at room temperature with 1 ml of 1% SDS, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA buffer, and the eluted DNA was precipitated with ice-cold ethanol. The nitrocellulose and DEAE-cellulose membranes were air-dried, and DNA retention was quantitated with a PhosphorImager
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