Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

Abstract

Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCEThe mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication.

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

“…Importantly, we have demonstrated that Rad51 binds to HPV31 genomes, with binding per genome increasing during productive replication. Knockdown of Rad51 and BRCA1, as well as inhibition of Rad51 activity using a small-molecule inhibitor, is sufficient to block productive replication but not viral genome maintenance, similarly to previously published studies after loss of ATM and MRN activity (4,32). Overall, these studies indicate that HPV may utilize ATM activity to facilitate recombination at viral genomes through Rad51 and BRCA1.…”

“…MRN facilitates the activation of ATM in response to DSBs and also functions downstream of ATM to facilitate HR repair, as mentioned above. We found that disruption of the MRN complex through depletion of Nbs1 abrogates productive replication of HPV31 and blocks the recruitment of Mre11 and Rad50, as well as Rad51, to viral genomes (32). Given the importance of ATM, the MRN complex, Rad51, and BRCA1 to DSB repair through HR, these results suggest that recombination may play a role in the amplification of HPV genomes.…”

“…(8,9). In support of this, multiple components of the ATM response, in addition to Rad51 and BRCA1, are required for productive replication, including the MRN complex (Mre11, Rad50, Nbs1) and Chk2 (4,32). ATM can stimulate recombination through two homology-directed pathways: Rad51-dependent strand invasion (referred to as HR) and single-strand annealing (SSA), both of which rely on a resection intermediate (49).…”

“…Five micrograms of resultant DNA was digested with BamHI (which does not cut the HPV31 genome) or HindIII (which linearizes the HPV31 genome) (New England BioLabs), separated on an 0.8% agarose gel for 15 h at 40 V, and subsequently transferred to a positively charged nylon membrane (Immobilon-Nyϩ; Millipore). 32 Plabeled linearized HPV31 genome was used as a probe.…”

“…In fact, DNA viruses such as simian virus 40 (SV40) (25), herpes simplex virus 1 (HSV-1) (26-28), Epstein-Barr virus (EBV) (29,30), and Kaposi's sarcoma-associated herpesvirus (KSHV) (31) have exhibited the use of replication-associated recombination in the synthesis of their genomes during infection. We recently demonstrated the importance of members of the DNA double-strand break recognition complex, MRN, in the HPV productive life cycle (32). MRN facilitates the activation of ATM in response to DSBs and also functions downstream of ATM to facilitate HR repair, as mentioned above.…”