Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

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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...

Section: Discussionmentioning

confidence: 99%

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

Mohni

Mastrocola

Bai

et al. 2011

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“…Due to the strong affinity of RPA32 for single-stranded DNA (102,107) and its function in recruiting the ATRIP-ATR complex to sites of DNA damage (7,26,106), it is not surprising that we and others (65,79) detected RPA32 in HSV-1-supported AAV2 RCs. In HSV-1-infected cells, RPA32 colocalizes with the HSV-1 major single-stranded DNA binding protein ICP8 (81) in HSV-1 RCs (82,93).…”

Section: Discussionmentioning

confidence: 99%

“…Besides viral helper factors, the fate of AAV2 replication also depends on cellular proteins. Recently, cellular proteins have been identified that interact with AAV2 Rep78/68 in adenovirus (Ad)-or HSV-1-supported AAV2 replication (63,65). Of these, the largest functional categories correspond to cellular proteins which are involved in DNA metabolism, including DNA replication, repair, and chromatin modification.…”

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confidence: 99%

“…During the course of infection, several small RCs rapidly expand and fuse to large structures, which displace the cellular chromatin and fill the entire cell nucleus (28,35,37,79,91). AAV2 RCs contain AAV2 proteins, as well as defined helper virus proteins and cellular proteins (3,35,63,65,75,79,90,91). Replicating AAV2 has inhibitory effects on both the host cell (9,41,68,71,73,74,100,101) and the helper virus (5,30,31,34,40,44,61,84,100).…”

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confidence: 99%

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Adeno-Associated Virus Type 2 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection

Vogel

Seyffert

Strasser

et al. 2012

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Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.A deno-associated virus type 2 (AAV2) is a small, nonenveloped parvovirus with a single-stranded DNA genome of 4.7 kb (52). In the absence of a helper virus, AAV2 establishes a latent infection characterized by site-specific integration of the viral genome into the AAVS1 site on human chromosome 19 (72). In the presence of a helper virus, AAV2 can replicate productively in the host cell nucleus. AAV2 DNA replication occurs at discrete sites in the nucleus, termed replication compartments (RCs). During the course of infection, several small RCs rapidly expand and fuse to large structures, which displace the cellular chromatin and fill the entire cell nucleus (28,35,37,79,91). AAV2 RCs contain AAV2 proteins, as well as defined helper virus proteins and cellular proteins (3,35,63,65,75,79,90,91). Replicating AAV2 has inhibitory effects on both the host cell (9,41,68,71,73,74,100,101) and the helper virus (5,30,31,34,40,44,61,84,100).One of the helper viruses for AAV2 replication is herpes simplex virus 1 (HSV-1) (14). The minimal HSV-1 helper factors for AAV2 replication from plasmid substrates include the helicaseprimase complex encoded by UL5, UL8, and UL52 and the major DNA binding protein ICP8 (3) (90). Besides viral helper factors, the fate of AAV2 replication also depends on cellular proteins. Recently, cellular proteins have been identified that interact with AAV2 Rep78/68 in adenovirus (Ad)-or HSV-1-supported AAV2 replication (63,65). Of these, the largest functional categories correspond to cellular proteins which are involved in DNA metabolism, including DNA replication, repair, and chromatin modification.There is accumulating evidence that the DNA damage response (DDR) pathways play central roles in viral replication (92). Control of DDR signaling may be a mechanism to prevent apoptosis and/or stop cell cycle progression (92)...

“…Thus, the role of viral exonucleases in DNA replication may be evolutionarily conserved among different dsDNA viruses. In addition, UL12 has been reported to play a role in resolving adeno-associated virus replication intermediates generated when HSV-1 is used as a helper virus (38). Viral and cellular recombination factors may collaborate to mediate viral recombination-dependent replication.…”

Section: Discussionmentioning

confidence: 99%

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

Balasubramanian

Bai

Buchek

et al. 2010

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The herpes simplex virus type 1 (HSV-1) alkaline nuclease, encoded by the UL12 gene, plays an important role in HSV-1 replication, as a UL12 null mutant displays a severe growth defect. The HSV-1 alkaline exonuclease UL12 interacts with the viral single-stranded DNA binding protein ICP8 and promotes strand exchange in vitro in conjunction with ICP8. We proposed that UL12 and ICP8 form a two-subunit recombinase reminiscent of the phage lambda Red ␣/␤ recombination system and that the viral and cellular recombinases contribute to viral genome replication through a homologous recombination-dependent DNA replication mechanism. To test this hypothesis, we identified cellular interaction partners of UL12 by using coimmunoprecipitation. We report for the first time a specific interaction between UL12 and components of the cellular MRN complex, an important factor in the ATM-mediated homologous recombination repair (HRR) pathway. This interaction is detected early during infection and does not require viral DNA or other viral or cellular proteins. The region of UL12 responsible for the interaction has been mapped to the first 125 residues, and coimmunoprecipitation can be abolished by deletion of residues 100 to 126. These observations support the hypothesis that cellular and viral recombination factors work together to promote efficient HSV-1 growth.The herpes simplex virus (HSV) genome replicates in the nucleus of an infected host cell, resulting in the production of longer-than-unit-length head-to-tail concatemers of viral DNA. Production of infectious virus requires the processing of concatemeric DNA into unit-length genomes by the packaging machinery followed by encapsidation into preassembled capsids (63). Several lines of evidence suggest that recombination plays a role in concatemer formation (69). Genomic inversions, proposed to occur through recombination, can be detected very early during infection, and these inversions require sequence homology (2,23,56). In addition, replication intermediates in HSV type 1 (HSV-1)-infected cells adopt a complex nonlinear structure that does not migrate in a pulsed-field gel, even after digestion with an enzyme that cuts once per unit length of the genome (1,33,51,71). Electron micrographs have revealed that replication intermediates are branched and contain Y-and X-shaped junctions (19,52). Furthermore, high levels of recombination have been reported not only between coinfecting viral strains (4, 17, 50, 62, 66) but also in plasmids containing repeated sequence elements (10, 11). These observations, taken together, are not consistent with a simple rolling circle mechanism of replication and suggest that, reminiscent of the bacteriophages T4 and lambda, HSV-1 utilizes a recombination-dependent replication mechanism to generate concatemeric viral DNA.We have previously reported that virus-encoded proteins are capable of participating in recombination events in vitro. The viral 5Ј-3Ј alkaline exonuclease (UL12) and the single-strand binding protein (ICP8) together mediate stra...