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J. Virol. doi:10.1128/JVI.00174-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Mutation in the Human Herpes Simplex Virus Type I UL52 Zinc Finger Motif Results in Defective Primase Activity but Can Recruit Viral Polymerase and Support Viral Replication Efficiently

Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT06030

^* To whom correspondence should be addressed. Email: weller{at}nso2.uchc.edu.

	Abstract

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase/primase complex consisting of UL5, UL8 and UL52. UL5 contains conserved helicase motifs, while UL52 contains conserved primase motifs, including a zinc finger motif. Although HSV-1 and HSV-2 UL52 contain a leucine residue at position 986, most other herpesvirus primase homologues contain a phenylalanine at this position. We constructed a HSV-1 UL52 L986F mutation and found that it can complement a UL52 null virus more efficiently than wild type. We thus predicted that the UL5/8/52 complex containing L986F mutation might posses increased primase activity; however, it exhibited only 25% of the WT level of primase activity. Interestingly, the mutant complex displayed elevated levels of DNA binding, ssDNA-dependent ATPase and helicase activities. This result confirms a complex interdependence between the helicase and primase subunits. We previously showed that primase defective mutants failed to recruit the polymerase catalytic subunit UL30 to prereplicative sites, suggesting that an active primase, or primer synthesis, is required for pol recruitment. Although L986F exhibits decreased primase activity, it can support efficient replication and recruit UL30 efficiently to replication compartments, indicating that a partially active primase is capable of recruiting polymerase. Extraction with detergents prior to fixation can extract nucleosolic proteins but not proteins bound to chromatin or the nuclear matrix. We showed that UL30 was extracted from replication compartments while UL42 remained bound, suggesting that UL30 maybe tethered to the replication fork by protein-protein interactions.

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