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.

Brian O Smith,Anthony W Oliver,Christian T Fontan,Apurva T Prabhakar,Claire D James,Renfeng Li,Dipon Das,Laurence H Pearl,Xu Wang,Andreas Wieland,Sarah H Glass,Matthew Day,Raymonde Otoa,Mary M Donaldson,Molly L Bristol,John Burgner,Iain M Morgan

doi:10.1128/mbio.01163-21

Abstract

ABSTRACTDuring the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication, and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A (E2 with serine 23 mutated to alanine) activates and represses transcription identically to E2-WT (wild-type E2), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Remarkably, S23A cells had a disrupted viral life cycle in organotypic raft cultures, with a loss of E2 expression and a failure of viral replication. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 and that this interaction is critical for the viral life cycle.

Highlights

Introduction of theS23A mutation into the human papillomavirus 16 (HPV16) genome resulted in a delay in immortalization, the resulting cells that grew out retained episomal viral genomes (Fig. 6)
Because TopBP1 binds to phosphorylated proteins, we investigated the ability of potential phosphorylation sites on E2 to mediate the interaction with TopBP1
To investigate the interaction between E2 and TopBP1 via serine 23, U2OS cells stably expressing E2-WT, E2-S23A, and E2S23D were generated, along with pcDNA empty vector plasmid control (Fig. 1B)

Summary

Introduction

S23A mutation into the HPV16 genome resulted in a delay in immortalization, the resulting cells that grew out retained episomal viral genomes (Fig. 6) These cells were grown on plastic with no feeder cells, and the S23A mutation had no effect on cell proliferation when the cells were grown in this manner. This is not reflective of any aspect of the HPV16 life cycle, and to determine the effect of the S23A mutation on this process, we submitted cells to organotypic rafting. The dramatic effects of the S23A mutation during organotypic rafting compared with cells cultured on plastic could be related to the interaction of the HPV16-positive cells with the collagen/fibroblast stroma and/or to the differentiation status of the cells.

Methods

Results

Conclusion