Contributions of nucleotide excision repair, DNA polymerase eta, and homologous recombination to replication of UV-irradiated herpes simplex virus type 1

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.

FIGURE 1.

Replication of UV-irradiated HSV-1 is impaired in cells deficient in nucleotide excision repair and DNA polymerase η. A, nonirradiated HSV-1 and UV-irradiated HSV-1 (UV/HSV-1) diluted 10⁵- and 10²-fold, respectively, and plated on wild type MRC5, XP12, deficient in the nucleotide excision repair XPA gene product, and XP30 cells, lacking DNA polymerase η. B, HSV-1 and UV/HSV-1 plaque efficiency on MRC5, XP12, XP30, and XP30 cells stably expressing DNA polymerase η as a GFP fusion protein, XP30eGFPη. C, plaque efficiency of HSV-1 and HSV-1 treated with calicheamicin (Clm/HSV-1) on MRC5, XP12, and XP30 cells. The numbers are the averages derived from three independent experiments.

FIGURE 2.

Growth curve analyses of HSV-1 and UV-irradiated HSV-1 on MRC5 and XP30 cells. A, cells were infected at a m.o.i. of 0.1 pfu/cell. B, cells were infected at a m.o.i. of 5 pfu/cell. Titers were determined by plaque assay on BHK-21 cells as described under “Experimental Procedures.” The numbers are the averages derived from three independent experiments.

FIGURE 3.

Effects of RNA interference against Rad51, Rad52 and Rad54 on HSV-1 and UV/HSV-1 replication in MRC5 cells. Cells were treated with siRNA for 72 h followed by infection with HSV-1 (top panel) and UV/HSV-1 (middle panel), for 24 h. Titers were determined by plaque assay on BHK-21 cells as described under “Experimental Procedures.” The numbers are the averages derived from three independent experiments. The bottom panel represents a Western blot analysis of Rad51, Rad52, and Rad54 following siRNA treatment.

FIGURE 4.

Repair of thymine dimers in UV-irradiated HSV-1 DNA. MRC5, XP30, and XP12 cells were infected with HSV-1 and UV/HSV-1 at a m.o.i. of 5 pfu/cell for the indicated times. Upper panel, total DNA was isolated and analyzed by immuno-dot blot using an anti-thymine dimer antibody (see “Experimental Procedures”). Lower panel, relative amounts of thymine dimers determined after quantification of the chemiluminescent signals on exposed films using a Fuji FLA-7000 bioimaging analyzer. Time p.i., time after infection.

FIGURE 5.

Effects of UV irradiation on HSV-1 gene expression. Cells were infected with HSV-1 and UV/HSV-1 at a m.o.i. of 5 pfu/cell for 12 h followed by Western blot analysis of the early gene product ICP8 and the late gene product glycoprotein C, in the precursor pgC and the mature gC forms (38). A, MRC5, XP12, and XP30 cells were used to examine effects of nucleotide excision repair and translesion synthesis by DNA polymerase η on gene expression. B, CSA and CSB cells were used to analyze effects of transcription-coupled nucleotide excision repair on gene expression.

FIGURE 6.

Model for repair of UV lesions during HSV-1 replication. A, transcription-coupled nucleotide excision repair (TCR) promotes expression of immediate early and early genes prior to DNA replication. Postreplication gap filling by DNA polymerase η is required for copying UV-damaged template DNA before late gene expression can start. Homologous recombination (HR) may act to repair double-stranded breaks resulting from incomplete replication. Global genome repair is likely to be active throughout the entire infectious cycle. B, postreplication gap filling by DNA polymerase η may create substrates for nucleotide excision repair (NER). C, simplified model illustrates how multiple rounds of DNA synthesis, initiated before DNA repair aided by DNA polymerase η has been completed, may result in double-stranded breaks. Because HSV-1 has three origins of DNA replication, multiple fragments may be generated especially at high m.o.i. These fragments may be substrates for homologous recombination acting to restore the physical integrity of the virus chromosome.