The JAK-STAT Transcriptional Regulator, STAT-5, Activates the ATM DNA Damage Pathway to Induce HPV 31 Genome Amplification upon Epithelial Differentiation

101

137

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.

mentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

Anacker

Gautam

Gillespie

et al. 2014

101

137

“…A proposed mechanism for the repression of transcription by E2 due to steric hindrance is based on the location of E2BS adjacent the TATA box, effectively blocking the binding of the TATA-binding protein (TBP) (Steger & Corbach, 1997). proposed to be essential for replication during vegetative amplification (Hong & Laimins, 2013). Topoisomerase II binding protein (TopBP1) and Breast Cancer 1 (BRCA1) were both found to interact with E2 and directly influence transcription/replication Kim, Lee, Gwan Hwang, Hwang, & Choe, 2003).…”

Section: E2mentioning

confidence: 99%

“…E7 is proposed to activate STAT5 phosphorylation which causes the activation of the ATM pathway (Hong & Laimins, 2013). E6 and E7 were found to independently activate caspases in differentiating cells (Moody, Fradet-Turcotte, Archambault, & Laimins, 2007).…”

Section: Papillomavirus Replicative Programmentioning

confidence: 99%

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Thomas

Androphy

2018

iii ACKNOWLEDGEMENTS I would like to thank my supervisor Elliot J. Androphy for his guidance, and encouragement over these many years. I truly appreciate your faith and continuous support.I also want to thank all members of the Androphy lab, for your guidance, critiques, and support.Thank you to my thesis committee, without whom none of this would be possible.Your guidance has been invaluable. Michael Klemsz, Suk-Hee Lee, Lindsey Mayo, and Andy Yu. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. v When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111. Human Papillomaviruses and DiseaseThere are over 200 HPVs that have been described and they manifest in a variety of ways; they can be asymptomatic, or they can result in benign lesions (papillomas) or progress to malignancy (de Villiers, Fauquet, Broker, Bernard, & zur Hausen, 2004). In the United States, HPV is the most common sexually transmitted infection and nearly all sexually active adults contract it at least once. E1The PV E1 protein is an essential viral replication factor (Lusky & Botchan, 1985;Ustav, Ustav, Szymanski, & Stenlund, 1991). E1 is the viral helicase and initiates replication by binding to specific sequences on the viral genome with the assistance of the E2 protein (Frattini & Laimins, 1994).There is sequence and functional homology between PV E1 and SV (simian virus) 40 Large T Antigen which allowed for many of the predictions concerning E2 shows differential affinity for the E2BS and it is proposed that at low levels, E2b...

“…In HPV-31, E7 was shown to activate STAT5 directly and E6 was not required (22). However, different HPV types are known to differ in the strategies employed to achieve the same state (24)(25)(26).…”

mentioning

confidence: 99%

PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

Thomas

Banks

2015

High-risk human papillomavirus (HPV) E6 proteins have a C-terminal PDZ binding motif through which they bind, and target for proteasome-mediated degradation, a number of PDZ-containing cellular targets. Recent studies have suggested that the RING-containing ubiquitin-protein ligase PDZRN3 might also be an HPV E6 target. In this analysis, we show that HPV-16 and HPV-18 E6 can target PDZRN3 in a PDZ-and proteasome-dependent manner and provide a connection between the HPV life cycle and differentiation-related STAT signaling. High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer, other anogenital cancers, and some head and neck cancers (1, 2). The E6 oncoproteins of high-risk HPV types have a C-terminal PDZ-binding motif (PBM) that is important for a normal viral life cycle and progeny production (for a review, see reference 3). PBM deletion causes viral genome integration, loss of replicative competence, and an increased potential for oncogenic transformation (4-8).E6 targets several PDZ-containing proteins (9-12), and recently, a proteomic screen of DNA tumor virus interactions (13) and an HPV-16 E6 C-terminal peptide screen of the human PDZome (14) Extracts of 293 cells transfected with pCDNA3.1 (lanes C) or pCDNA-FLAG-PDZRN3A (lanes P) were incubated with GST, GST-HPV18E6, or GST-HPV18E6T156E, as indicated.Afterwashing,theboundproteinswereanalyzedbyaWesternblotassayprobedwithanti-PDZRN3andanti-GSTantibodiestoconfirmequalloadingofGSTproteins. (C) HPV-18 E6-induced degradation of PDZRN3 is proteasome dependent. A plasmid expressing FLAG-tagged PDZRN3A was transfected into 293 cells alone or with an HPV-18 E6-expressing plasmid. After overnight incubation, the cells were treated with the proteasome inhibitors carbobenzoxy-Leu-Leu-leucinal (CBZ) and N-acetyl-L-leucyl-L-leucyl-L-norleucinal for 3 h before harvesting. Cell extracts were analyzed by Western blotting with anti-FLAG antibody; ␤-galactosidase was included as a control for transfection efficiency.