Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.Herpes simplex virus type 1 (HSV-1) is a linear doublestranded DNA virus that replicates in the nucleus of the infected cell. Viral DNA synthesis takes place within globular domains called replication compartments (55), which contain the seven essential viral DNA replication proteins: the originbinding protein (UL9), the single-stranded-DNA-binding protein (UL29 or ICP8), the helicase-primase heterotrimer (UL5/ UL8/UL52), the viral polymerase (UL30), and its processivity factor (UL42) (reviewed in reference 75). Due to the lack of a reconstituted in vitro replication system, it has not been possible to determine whether cellular proteins are also involved in HSV-1 DNA replication.Several lines of evidence indicate that the process of HSV-1 DNA replication is linked to recombination. For example, recombination is a frequent event within the HSV-1 genome as well as between infecting genomes and is stimulated on HSV-1 infection (14,15,20,45,59,71,72). Furthermore, newly replicated DNA is larger than unit length and adopts a highly complex, possibly branched structure (6,38,60,61,64), the formation of which is presumed to require recombination (reviewed in reference 37). Newly replicated DNA has also undergone genomic inversion (4,28,60,81). Thus, the properties of replicating DNA indicate a lik...

Like other DNA viruses, herpes simplex virus type 1 (HSV-1) interacts with components of the cellular response to DNA damage. For example, HSV-1 sequesters endogenous, uninduced, hyperphosphorylated RPA (replication protein A) away from viral replication compartments. RPA is a ssDNA-binding protein that signals genotoxic stress through the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway. The sequestration of endogenous hyperphosphorylated RPA away from replicating viral DNA suggests that HSV-1 prevents the normal ATRsignaling response. In this study we examine the spatial distribution of endogenous hyperphosphorylated RPA with respect to ATR, its recruitment factor, ATRIP, and the cellular dsDNA break marker, ␥ ␥H2AX, during HSV-1 infection. The accumulation of these repair factors at DNA lesions has previously been identified as an early event in signaling genotoxic stress. We show that HSV-1 infection disrupts the ATR pathway by a mechanism that prevents the recruitment of repair factors, spatially uncouples ATRIP from ATR and sequesters ATRIP and endogenous hyperphosphorylated RPA within virus-induced nuclear domains containing molecular chaperones and components of the ubiquitin proteasome. The HSV-1 immediate early protein ICP0 is sufficient to induce the redistribution of ATRIP. This is the first report that a virus can disrupt the usually tight colocalization of ATR and ATRIP.

SummaryIn many organisms the processes of DNA replication and recombination are closely linked. For instance, in bacterial and eukaryotic systems, replication forks can become stalled or damaged, in many cases leading to the formation of double stranded breaks. Replication restart is an essential mechanism in which the recombination and repair machinery can be used to continue replication after such a catastrophic event. DNA viruses of bacteria such as lambda and T4 also rely heavily on DNA recombination to replicate their genomes and both viruses encode specialized gene products which are required for recombinationdependent replication. In this review, we examine the linkage between replication and recombination in the eukaryotic pathogen, Herpes Simplex Virus Type 1 (HSV-1). The evidence that recombination plays an intrinsic role in HSV-1 DNA replication and the infection process will be reviewed. We have recently demonstrated that HSV-1 encodes two proteins which may be analogous to the lambda phage recombination system, Reda and b. The HSV-1 alkaline nuclease, a 5' to 3' exonuclease, and ICP8, a single stranded DNA binding protein, can carry out strand annealing reactions similar to those carried out by the lambda Red system. In addition, evidence suggesting that host recombination proteins may also be important for HSV-1 replication will be reviewed. In summary, it is likely that HSV-1 infection will require both viral and cellular proteins which participate in various pathways of recombination and that recombination-dependent replication is essential for the efficient replication of viral genomes.

Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains.Productive herpes simplex virus type 1 (HSV-1) infection results in the formation of globular domains in the nucleus, called replication compartments, in which viral DNA replication and cleavage and packaging of the viral genome occur (40, 54). The HSV-1 genome encodes seven essential replication proteins, all of which are localized to replication compartments in productively infected cells: the single-stranded DNA binding protein ICP8, the heterotrimeric helicase-primase complex UL5/UL8/UL52, the origin-binding protein UL9, and the heterodimeric polymerase complex UL30/UL42 (53, 66). Replication compartments form via a series of protein scaffold intermediate structures called prereplicative sites that contain HSV-1 replication proteins and cellular proteins (7,13,37,42,45,54,68,69). In this paper we confirm a previous report (8) that functional ICP8 protein is required for the formation of prereplicative sites.In order to identify subassemblies or stages in the formation of replication compartments, it has been convenient to freeze the progression either by infection with viruses bearing mutations in replication proteins or by the use of pharmacological agents that inhibit viral DNA synthesis (see Fig. 1) (7, 68). Stage I is defined by the presence of nuclear domains (NDs) that resemble ND10 (promyelocytic leukemia protein [PML] bodies or PML oncogenic domains) and the absence of ICP8 as detected by immunofluorescence microscopy. Work from Everett and colleagues suggests that ND10s that are detected during stage I of infection represent reformed ND10-like foci that have been recruite...

Variability in viral load measurements using nucleic acid amplification techniques (NAT) has a significant impact on the management of Epstein-Barr virus (EBV)-associated diseases, and has highlighted a need for standardisation of these measurements. The aim of this collaborative study was to evaluate the suitability of a range of candidate reference materials to harmonise EBV viral load measurements in a wide range of NAT assays. Candidate materials included lyophilised and liquid whole virus preparations of the EBV B95-8 strain, and preparations of Namalwa and Raji cells. Variability between the individual laboratory mean estimates for each candidate was 2.5 log10 copies/mL. The agreement between laboratories was improved when the potency of each candidate was expressed relative to the lyophilised B95-8 preparation. The results of the study indicate the suitability of this candidate as the 1st WHO International Standard for EBV for NAT. It was established in October 2011 by the WHO's Expert Committee on Biological Standardisation with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/260). It is intended to be used for the calibration of secondary reference materials, used in EBV NAT assays, in IU, thereby improving the comparability of patient viral load measurements.