DNA Mismatch Repair Proteins Are Required for Efficient Herpes Simplex Virus 1 Replication

Abstract

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of its human host cell and is known to interact with many cellular DNA repair proteins. In this study, we examined the role of cellular mismatch repair (MMR) proteins in the virus life cycle. Both MSH2 and MLH1 are required for efficient replication of HSV-1 in normal human cells and are localized to viral replication compartments. In addition, a previously reported interaction between MSH6 and ICP8 was confirmed by coimmunoprecipitation and extended to show that UL12 is also present in this complex. We also report for the first time that MLH1 associates with ND10 nuclear bodies and that like other ND10 proteins, MLH1 is recruited to the incoming genome. Knockdown of MLH1 inhibits immediate-early viral gene expression. MSH2, on the other hand, which is generally thought to play a role in mismatch repair at a step prior to that of MLH1, is not recruited to incoming genomes and appears to act at a later step in the viral life cycle. Silencing of MSH2 appears to inhibit early gene expression. Thus, both MLH1 and MSH2 are required but appear to participate in distinct events in the virus life cycle. The observation that MLH1 plays an earlier role in HSV-1 infection than does MSH2 is surprising and may indicate a novel function for MLH1 distinct from its known MSH2-dependent role in mismatch repair.Herpes simplex virus 1 (HSV-1) is a large double-stranded DNA virus that replicates in the nucleus of the host cell. Although cis-and trans-acting functions involved in HSV-1 replication in infected cells have been identified (69), the mechanism of HSV-1 DNA replication remains poorly understood. The appearance of viral genomes in the nucleus might be expected to induce a cellular DNA damage response, and it is now clear that HSV-1 has evolved a complex relationship with the host DNA damage response pathways (33,48,60,66,70,71). During infection, cellular factors that are beneficial to the virus are hijacked and recruited, while other factors and pathways are degraded or inactivated. Previous work from our group and others has shown that the ATR (ATM-and Rad3-related) and DNA-PK (DNA-dependent protein kinase) DNA damage-sensing kinases are attenuated in HSV-1-infected cells, while the ATM (ataxia-telangiectasia-mutated) kinase is activated (31,34,48,50). In addition, other components of the DNA damage repair and response pathways also appear to be beneficial to the virus and are utilized during viral replication (35). Many host cellular DNA damage proteins are known to localize to viral replication compartments (33,48,60,66,71). Furthermore, in a recent proteomic analysis of replication compartments, several DNA repair proteins were identified as potential interacting partners of the viral single-stranded DNA (ssDNA) binding protein ICP8 (66). One such potential interacting partner, the DNA mismatch repair (MMR) protein MSH2, was shown to localize to the replication compartments in infected cells (66).The DNA MMR system is highly c...

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

“…7D). The observation that the recruitment of Rad9 to replication compartments is independent of RPA may indicate that Rad9 is recruited by a viral protein or by interaction with one of its other cellular interacting partners, such as MLH1 (64), which is also in replication compartments (4).…”

“…Immunofluorescence (IF) analysis was performed as described previously (4,6,32). Briefly, cells adhering to glass coverslips were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100.…”

“…The pLKO.1 system was used to package lentiviruses and to deliver shRNA into target cells as previously described (4,6). Lentiviruses expressing short hairpin RNA targeting GFP (shGFP), shATR, shATRIP-1, and shATRIP-2 were described previously (6).…”

“…After entry into the nucleus, viral DNA is recognized by cellular homeostatic mechanisms, including the ND10 components PML, sp100, and hDaxx (1,2), as well as cellular double-strand break (DSB) repair proteins (3,4). It is becoming increasingly clear that the ND10 and DSB repair pathways represent intrinsic cellular antiviral defense strategies, and both are counteracted by the viral E3 ubiquitin ligase ICP0 (1,3).…”

“…It is becoming increasingly clear that the ND10 and DSB repair pathways represent intrinsic cellular antiviral defense strategies, and both are counteracted by the viral E3 ubiquitin ligase ICP0 (1,3). Viral DNA replication itself also elicits a cellular DNA damage response and results in the specific recruitment of cellular DNA repair proteins to sites of viral DNA replication (4)(5)(6)(7)(8)(9).…”