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

Abstract

The papillomavirus (PV) 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. Posttranslational modifications (PTMs) of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine 111 (K111) as a target of p300 acetylation in bovine PV (BPV). The di-lysines at 111 and 112 are conserved in almost all papillomaviruses. We pursued a mutational approach to query the functional significance of lysine in human PV (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. While the replication-defective E2-K111R mutant recruited E1 to the viral replication origin, surprisingly, unwinding of the duplex DNA did not occur. In contrast, the K111 glutamine (K111Q) mutant increased origin melting and stimulated replication compared to wild-type E2. These experiments reveal a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.IMPORTANCE HPV is one of the most common sexually transmitted infections in the United States. Over 200 HPVs have been described, and they manifest in a variety of ways; they can be asymptomatic or can result in benign lesions (papillomas) or progress to malignancy. Although 90% of infections are asymptomatic and resolve easily, HPV16 and -18 alone are responsible for 70% of all cervical cancers, which are almost entirely caused by HPV infection. Interestingly, 60 to 90% of other cancers have been linked to HPV. The goal of this research is to further elucidate the mechanisms that regulate and mediate viral replication.

Keywords: acetylation; lysine; papillomavirus; viral replication.

FIG 1

K111 is necessary for transient viral replication. Mutations of E2 lysine 111 (K111) alter transient replication. (A) Sequence alignment shows the highly conserved K111. (B) C33A cells were cotransfected with BPV1 E2-K111 mutants, pFLORI-BPV1, and pRRL and (C) C33A cells were cotransfected with HPV31 E2-K111 mutants. E2 mutants were cotransfected with pFLOri31. The luminescence was normalized to Renilla luciferase and the respective background E2 luminescence. Each of the samples was normalized to its respective control without E1 to cancel out any background effect. (Inset) The effect of K111R mutation on cell viability and cell cycle was determined by flow cytometry. (D) 293TT cells were cotransfected with HPV31 FLAG-E2 and HA-E1. E1 protein pulldown was conducted using 12CA5 antibody and blotted with HA7 and M2 antibodies. *, P < 0.05; **, P < 0.005.

FIG 2

K111 is not required for nuclear localization of E2. C33A cells and NIKs were transfected with the HPV31 E2 wild type (wt) and the K111 mutants. The cells were stained with HA7 48 h posttransfection and analyzed by immunofluorescence for E2 (red) and DAPI (blue).

FIG 3

HPV31 E2 lysine 111 (K111) mutations repress transcription. C33A cells were cotransfected with 31-Fluc and HPV31 FLAG-tagged E2 wild type (wt) or K111 mutants. The amount of E2 at the origin was determined by ChIP using LCR4 primers (A). C33A cells were cotransfected with 31-Fluc and HPV31 FLAG-E2 wild type or K111 mutants. Repression was measured by luminometry and is shown as a fraction of baseline transcription (B). HeLa cells were transfected with HPV31 HA-E2 wild type and harvested after 48 h, and E6 transcripts were quantified by qPCR (C). *, P < 0.05.

FIG 4

HPV31 E2-K111 mutants maintain interactions with cellular factors. (A) 293TT cells were cotransfected with HPV31 FLAG-E2, and plasmids expressing BRD4 fragments CTM, BID, and PDID. BRD4 proteins were pulled down using glutathione S-transferase (GST) antibody and blotted with GST and M2 antibodies. (B) 293TT cells were cotransfected with HPV31 FLAG-E2 and HA-GPS2. HPV31 E2 protein pulldown was executed with M2 antibody and blotted with HA7 and M2 antibodies. Relative GPS2 binding was calculated by densitometry and presented graphically. (C) 293TT cells were transfected with HPV31 FLAG-E2 followed by E2 protein immunoprecipitation with M2 antibody. The amounts of endogenous TopBP1 coimmunoprecipitated were determined by blotting with anti-TopBP1 and anti-M2 antibodies, and relative binding was calculated by densitometry and represented graphically.

FIG 5

E1 protein complexes with and is recruited to the replication origin by the E2-K111 mutants. (A) C33A cells were cotransfected with PFLORI31, HPV31 HA-E2, and FLAG-E1. ChIP for E1 was conducted with M2 antibody and LCR4 primers. (B) HeLa cells were transfected with HPV31 FLAG-E1 and HA-E2, and the presence of E2 at the HeLa origin was determined by ChIP. *, P < 0.05; **, P < 0.005.

FIG 6

The K111R mutant reduces unwinding of the replication origin. C33A cells were transfected with FLAG-E1, FLAG-E2, or PFLORI31. Unwinding at origin DNA was analyzed by FAIRE assay using HPV31 LCR primers LCR3 and LCR4 (A). (B) ChIP for RPA protein at the LCR used anti-RPA70 antibody and LCR4 primers. *, P < 0.05 by one-way ANOVA compared to the wild type in the absence of E1; #, P < 0.05 by one-way ANOVA compared to wild-type E2 plus E1.