Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

Abstract

Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle. IMPORTANCE HPV16 is the major human viral carcinogen, responsible for around 3 to 4% of all cancers worldwide. Our understanding of how the viral replication machinery interacts with host factors to control/activate the DNA damage response to promote the viral life cycle remains incomplete. Recently, we demonstrated a SIRT1-WRN axis that controls HPV16 replication, and here we demonstrate that this axis persists in clinical cervical lesions induced by HPV16. Here, we describe the effects of WRN depletion on cellular differentiation with or without HPV16; WRN depletion results in enhanced proliferation and DNA damage irrespective of HPV16 status. Also, WRN is a restriction factor for the viral life cycle since replication is disrupted in the absence of WRN. Future studies will focus on enhancing our understanding of how WRN regulates viral replication. Our goal is to ultimately identify cellular factors essential for HPV16 replication that can be targeted for therapeutic gain.

CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

“…While the growth of the cells was not different when grown on plastic, this does not represent a physiologically relevant part of the HPV16 life cycle. To investigate whether mutation of serine 23 disrupted the HPV16 life cycle, in addition to attenuating initial immortalization, we carried out organotypic raft cultures, as we have described previously ( 92 , 93 ). Figure 7A gives a representative image from hematoxylin and eosin (H&E) staining of the organotypic rafts from donor A containing the wild-type (WT) HPV16 and HPV16 S23A genomes ( Fig.…”

“…Keratinocytes were differentiated via organotypic raft culture as described previously ( 81 , 93 ). Briefly, cells were seeded onto type 1 collagen matrices containing J2 3T3 fibroblast feeder cells.…”

A Critical Role for p53 during the HPV16 Life Cycle

“…Keratinocytes were differentiated via organotypic raft culture as described previously ( 62 , 76 , 79 ). Briefly, cells were seeded onto type 1 collagen matrices containing J2 3T3 fibroblast feeder cells.…”

RecQ Helicase Somatic Alterations in Cancer

“…Treatment with siRNA for WRN increased sensitivity of cancer cells to CPT-11, a topoisomerase I inhibitor. Decreased WRN mRNA expression negatively correlated with cervical cancer progression and WRN protein regulates the life cycle of viral carcinogen human papillomavirus 16 (HPV-16), linked with causing cervical and oropharyngeal cancers ( James et al, 2020 ).…”