Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

Abstract

, virus replication was reduced 10 6 -, 400-, and 100-fold, respectively. In DNA polymerase mutant cells HSV-1 plaque efficiency was reduced 10 4 -fold. Furthermore, DNA polymerase 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 may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.Herpes simplex virus has a linear double-stranded genome, which, upon entry into the eukaryotic nucleus, circularizes and becomes replicated by a replisome consisting of six virus-encoded proteins (1-4). The herpes simplex virus replisome is composed of a DNA polymerase made up from the UL30 and UL42 gene products, a helicase-primase complex encoded by the UL5, UL8, and UL52 genes and a single-stranded DNAbinding protein ICP8, which is a product of the UL29 gene (4, 5). The replisome is loaded on the origins of replication oriS and oriL by a sequence-specific superfamily II DNA helicase termed OBP or UL9 protein (6 -9), and it is capable of processive leading strand DNA synthesis coupled to discontinuous synthesis of lagging strand intermediates (5). Processive polymerization is dependent on the UL42 protein, which is a monomer folded as the proliferating cell nuclear antigen (PCNA) 2 protomer but with no sequence similarity to the cellular processivity factor (10). The UL30 subunit is a proofreading family B DNA polymerase (11). It appears that the overall replication fidelity is high, and existing genetic variability may be created by recombination between a limited number of strains rather than random replication errors (12)(13)(14). The contribution of the DNA polymerase to replication fidelity has been thoroughly examined, but cellular mechanisms contributing to the genetic stability of herpesviruses have, with few exceptions, not been extensively looked at (15-18). It has been noted that several cellular repair proteins co-localize with viral replication proteins in a limited number of replication foci (19 -21). In cells, genomic stability is maintained by an intricate interplay among the replication machinery, repair proteins, and factors controlling the cell cycle (22,23). Viruses are known to interact and interfere with these mechanisms to promote their own replication...

Physical Interaction between the Herpes Simplex Virus Type 1 Exonuclease, UL12, and the DNA Double-Strand Break-Sensing MRN Complex

“…DSBs are sensed by the MRN complex to signal ATM activation or by Ku70/Ku86 to activate DNAPKcs. As mentioned above, ATM targets are phosphorylated in infected cells (30,55,55,68), and virus yields are decreased in cells deficient in ATM, WRN, Chk2, Mre11, and Rad51 (29,30,37,60), suggesting that these proteins may play a positive role in HSV infection. On the other hand, HSV-1 infection results in the degradation of DNA-PKcs in many cell types (28,40), and viral yields increase severalfold in cells deficient for DNA-PKcs and Ku70 (40,60), indicating that at least some components of NHEJ may be inhibitory for viral growth.…”

“…Several cellular proteins known to participate in host damage responses are recruited to replication compartments (15,30,55,60,65,68) and are required for efficient virus production (29,30,37,60). The MRN complex, consisting of Mre11, Rad50, and Nbs1, is particularly important in the normal cellular damage response, as it acts as a sensor of double-strand breaks (DSBs) and is recruited to the site of DNA damage, thereby signaling activation of at least one of the phosphatidylinositol 3-kinase (PI3 kinase)-like kinases (PIKKs), ATM (ataxia-telangiectasia mutated) (24,30,41,42,70).…”

The Lytic Phase of Epstein-Barr Virus Requires a Viral Genome with 5-Methylcytosine Residues in CpG Sites

“…Several lines of evidence support our view. First is PCNA, which is critical for tethering the cellular DNA methyltransferase DNMT1 to newly replicating, hemi-methylated DNA but does not participate in herpesviral DNA amplification (36). Second, DNMT1 levels are regulated with the cell cycle and induced upon S-phase entry (48).…”

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

“…In addition, various DNA repair mechanisms may contribute to viral genome maintenance. In this regard, a recent study showed that the replication of HSV-1 containing UV-induced lesions is reduced to various levels in cells with defects in nucleotide excision repair (XP-A), translesion synthesis (pol ), transcription-coupled nucleotide excision repair (CS-A and CS-B), and homologous recombination (Rad 51, Rad 52 and Rad 54), with the most pronounced effect contributed by a deficiency in XP-A, decreasing virus production 10 6 -fold (37). Base excision repair (BER) is a further mechanism that contributes to genome maintenance by removing unusual bases from the DNA and to repair apurinic/ apyrimidinic (AP) sites resulting from spontaneous base loss (reviewed in Ref.…”