Abstract

The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.

Highlights

  • Ing strand DNA synthesis coupled to discontinuous synthesis of lagging strand intermediates [5]

  • As a first step toward clarifying the role of cellular repair systems during herpesvirus replication, we have looked at repair of UV-induced lesions in herpes simplex virus type 1 (HSV-1) DNA and how they affect the progression of the viral replication cycle

  • Nucleotide Excision Repair, Translesion Synthesis, and Homologous Recombination Contribute to Repair of UV-damaged HSV-1 DNA—To monitor repair of UV-damaged HSV-1, we used unirradiated cell lines mutated in essential repair proteins

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Summary

EXPERIMENTAL PROCEDURES

Viruses, and Plaque Assays—MRC5, XP12 (XPA), XP30 (XPV), and XP30eGFP␩, which is stably transfected with a plasmid encoding the wild type DNA polymerase ␩, were kindly provided by Alan R. For production of calicheamicin/HSV-1, HSV-1 viruses were mock-treated or treated with 0.1 and 1 ␮M calicheamicin (kindly provided by Ola Hammarsten, University of Gothenburg, Sweden) for 1 h on ice, followed by overnight incubation with 1 mM dithiothreitol on ice. Growth Curve Analysis—MRC5 and XP30 monolayers were infected with HSV-1 and UV/HSV-1 at a m.o.i. of 5 or 0.1 plaque-forming units (pfu)/cell at 37 °C. For detection of ICP8 and glycoprotein C, the indicated cell monolayers were infected with HSV-1 or UV/HSV-1 at an m.o.i. of 5 pfu/cell for 12 h followed by Laemmli lysis buffer treatment and Western blot analysis, using rabbit polyclonal antibodies to HSV-1 ICP8 and mouse monoclonal antibodies to HSV-1 glycoprotein C (kindly provided by Tomas Bergstrom, University of Gothenburg, Sweden).

RESULTS
DISCUSSION
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