PMID- 27795438 OWN - NLM STAT- MEDLINE DCOM- 20170515 LR - 20210109 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 91 IP - 1 DP - 2017 Jan 1 TI - The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment. LID - 10.1128/JVI.01853-16 [doi] LID - e01853-16 AB - In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2(Y131A)) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2(Y131A) showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2(WT)), the chromatin-bound pool of E2(Y131A) was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2(Y131A) occurred in mid-S phase. A similar alteration between the subcellular pools of the E2(WT) protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2(Y131A) genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. IMPORTANCE: Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection is a risk factor for cancer development and is partly achieved by the attachment of viral DNA to cellular chromatin during cell division. The HPV E2 protein plays a critical role in this tethering by binding simultaneously to the viral genome and to chromatin during mitosis. We previously showed that the cellular DNA helicase ChlR1 is required for loading of the bovine papillomavirus E2 protein onto chromatin during DNA synthesis. Here we identify a mutation in HPV16 E2 that abrogates interaction with ChlR1, and we show that ChlR1 regulates the chromatin association of HPV16 E2 and that this virus-host interaction is essential for viral episome maintenance. CI - Copyright (c) 2016 Harris et al. FAU - Harris, Leanne AU - Harris L AD - Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom. FAU - McFarlane-Majeed, Laura AU - McFarlane-Majeed L AD - Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom. FAU - Campos-Leon, Karen AU - Campos-Leon K AD - Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom. FAU - Roberts, Sally AU - Roberts S AD - Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom. FAU - Parish, Joanna L AU - Parish JL AD - Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom j.l.parish@bham.ac.uk. LA - eng GR - 11976/CRUK_/Cancer Research UK/United Kingdom GR - MR/N023498/1/MRC_/Medical Research Council/United Kingdom PT - Journal Article DEP - 20161216 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 0 (Chromatin) RN - 0 (DNA, Viral) RN - 0 (DNA-Binding Proteins) RN - 0 (E2 protein, Human papillomavirus type 16) RN - 0 (Oncogene Proteins, Viral) RN - 9007-49-2 (DNA) RN - EC 3.6.4.- (DNA Helicases) RN - EC 3.6.4.13 (DDX11 protein, human) RN - EC 3.6.4.13 (DEAD-box RNA Helicases) SB - IM MH - Chromatin/chemistry/metabolism MH - DEAD-box RNA Helicases/*genetics/metabolism MH - DNA/genetics/metabolism MH - DNA Helicases/*genetics/metabolism MH - DNA, Viral/*genetics/metabolism MH - DNA-Binding Proteins/*genetics/metabolism MH - Gene Dosage MH - Gene Silencing MH - *Genome, Viral MH - Host-Pathogen Interactions MH - Human papillomavirus 16/*genetics/metabolism MH - Humans MH - Keratinocytes/metabolism/virology MH - Mitosis MH - Models, Molecular MH - Mutation MH - Oncogene Proteins, Viral/*genetics/metabolism MH - Plasmids/genetics/metabolism MH - Primary Cell Culture MH - Protein Binding MH - Protein Structure, Secondary MH - S Phase Cell Cycle Checkpoints MH - Transcriptional Activation PMC - PMC5165203 OTO - NOTNLM OT - DNA helicase OT - episome OT - papillomavirus OT - persistence OT - virus-host interaction EDAT- 2016/11/01 06:00 MHDA- 2017/05/16 06:00 PMCR- 2016/12/16 CRDT- 2016/11/01 06:00 PHST- 2016/09/14 00:00 [received] PHST- 2016/10/07 00:00 [accepted] PHST- 2016/11/01 06:00 [pubmed] PHST- 2017/05/16 06:00 [medline] PHST- 2016/11/01 06:00 [entrez] PHST- 2016/12/16 00:00 [pmc-release] AID - JVI.01853-16 [pii] AID - 01853-16 [pii] AID - 10.1128/JVI.01853-16 [doi] PST - epublish SO - J Virol. 2016 Dec 16;91(1):e01853-16. doi: 10.1128/JVI.01853-16. Print 2017 Jan 1.