Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA)-binding protein, is a heterotrimeric complex composed of three tightly associated subunits of 70, 32, and 14 kDa (referred as to RPA70, RPA32, and RPA14, respectively) that is essential for DNA replication, recombination, and all major types of DNA repair (4). RPA participates in such diverse pathways through its ability to interact with DNA and numerous proteins involved in its processing. During DNA replication, RPA associates with ssDNA at forks and facilitates nascent-strand DNA synthesis by replicative DNA polymerases localized at replication foci during S phase. Under DNA-damaging conditions, RPA binds to ssDNA at damaged sites and interacts with repair and recombination components to process double-strand DNA breaks (DSBs) and other lesions (6, 14, 21, 32, 38, 41).RPA undergoes both DNA damage-independent and -dependent phosphorylation on the N-terminal 33 residues of RPA32. Unstressed cell cycle-dependent phosphorylation occurs during the G₁/S-phase transition and in M phase, primarily at the conserved cyclin-CDK phosphorylation sites of Ser-23 and Ser-29 in the N terminus of the RPA32 subunit (13, 15). In contrast, stress-induced hyperphosphorylation of RPA is much more extensive. Nine potential phosphorylation sites within the N-terminal domain of RPA32, Ser-4, Ser-8, Ser-11/Ser-12/Ser-13, Thr-21, Ser-23, Ser-29, and Ser-33, in response to DNA-damaging agents, have been suggested (33, 54). Although this region of RPA32 is not required for the ssDNA-binding activity of RPA (5, 22), a phosphorylation-induced subtle conformation change in RPA, resulting from altered intersubunit interactions, regulates the interaction of RPA with both interacting proteins and DNA (30). The hyperphosphorylated form of RPA32 is unable to localize to replication centers in normal cells, while binding to DNA damage foci is unaffected (46). Therefore, RPA phosphorylation following damage is thought to both prevent RPA from catalyzing DNA replication and potentially serve as a marker to recruit repair factors to sites of DNA damage. RPA localizes to nuclear foci where DNA repair is occurring after DNA damage and is essential for multiple DNA repair pathways, participating in damage recognition, excision, and resynthesis reactions (4, 56).Mammalian cells can repair DSBs by homologous recombination (HR) or by nonhomologous end joining. HR is an accurate repair process, the first step of which is the resection of the 5′ ends of the DSB to generate 3′ ssDNA overhangs. This reaction is carried out by the Mre11/Rad50/Nbs1 (MRN) complex, which not only functions as a damage sensor upstream of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) activation but also plays a role in DSB repair (4). RPA and members of the RAD52 epistasis group of gene products, such as Rad51, Rad52, and Rad54, bind to the resulting 3′ ssDNA strands and form a helical, nucleoprotein filament that facilitates the invasion of a damaged DNA strand into the homologous double-stranded DNA partner. The human Rad51 protein is a structural and functional homolog of the Escherichia coli RecA protein, which promotes homologous pairing and strand transfer reactions in vitro. Both Rad51 and Rad52 bind specifically to the terminal regions of tailed duplex DNA, the substrate thought to initiate recombination in vivo. Furthermore, nucleoprotein filaments of Rad51, formed on tailed DNA, catalyze strand invasion of homologous duplex DNA in a reaction that is stimulated by Rad52 and RPA (3).Epstein-Barr virus (EBV) is a human herpesvirus that infects B lymphocytes, inducing their continuous proliferation. In B-lymphoblastoid cell lines, there is no production of virus particles, which is termed latent infection (52). Reactivation from latency is characterized by the expression of lytic genes, and one of the first detectable changes is the expression of the BZLF1 immediate-early gene product, which trans-activates viral promoters (16), leading to an ordered cascade of viral early and late gene expression. This lytic EBV DNA replication occurs in discrete sites in nuclei, called replication compartments, in which seven viral replication proteins are assembled (44). The viral genome is amplified several hundredfold by the viral replication machinery and is thought to generate highly branched replication intermediates through HR coupled with viral DNA replication (48). With the progression of lytic replication, the replication compartments become larger and appeared to fuse to form large globular structures that eventually filled the nucleus at late stages of infection (8, 45).We previously isolated latently EBV-infected Tet-BZLF1/B95-8 cells in which the exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter, leading to a highly efficient induction of lytic replication (28). Using this system, we have demonstrated that the induction of the EBV lytic program results in the inhibition of replication of cellular DNA in spite of the replication of viral DNA (28) and elicits a cellular DNA damage response, with the activation of the ATM-Chk2-p53 DNA damage transduction pathway (29). The DNA damage sensor MRN complex and phosphorylated ATM are recruited and retained in viral replication compartments (29).Here we report that RPA32 is extensively phosphorylated after EBV lytic replication is induced, with the phosphorylation sites in accordance with those for ATM. Phosphorylated RPA, Rad51, and Rad52, which are involved in HR repair (HRR), are recruited and retained in viral replication compartments as well as the MRN complex. Furthermore, DSBs could be demonstrated to occur during viral genome synthesis in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference (RNAi) knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that HR and/or repair of viral DNA genome might occur, coupled with DNA replication, to facilitate viral genome synthesis.     

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.     

Replication of Viral Deoxyribonucleic Acid and Breakdown of Cellular Deoxyribonucleic Acid in Epstein-Barr Virus Infection

Infection of Raji cells with Epstein-Barr virus (EBV) causes suppression of cellular deoxyribonucleic acid (DNA) synthesis and fragmentation of the cellular DNA. About 1,000 copies of EBV DNA of normal size (about 5 x 10(7) daltons in a single strand, as shown in an alkaline gradient) are synthesized per cell.     

Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication.     

The S2 Gene of Equine Infectious Anemia Virus Is Dispensable for Viral Replication In Vitro

Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAV_UK. Various EIAV_UK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAV_UK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.