GENOME REPLICATION AND REGULATION OF VIRAL GENE EXPRESSION

crossm The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2 Dipon Das,a Nathan Smith,a Xu Wang,a Iain M. Morgana,b VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, Virginia, USAa; VCU Massey Cancer Center, Richmond, Virginia, USAb

ABSTRACT Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle.

Received 19 January 2017 Accepted 23 February 2017 Accepted manuscript posted online 8 March 2017 Citation Das D, Smith N, Wang X, Morgan IM. 2017. The deacetylase SIRT1 regulates the replication properties of human papillomavirus 16 E1 and E2. J Virol 91:e00102-17. https://doi .org/10.1128/JVI.00102-17. Editor Lawrence Banks, International Centre for Genetic Engineering and Biotechnology Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Iain M. Morgan, [email protected].

IMPORTANCE HPV are causative agents in a number of human diseases, and cur-

rently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease. KEYWORDS C33a cells, CRISPR/Cas9, DNA replication, E1, E2, papillomavirus, SIRT1

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uman papillomaviruses (HPV) are causative agents in a number of human diseases, ranging from anogenital and oropharyngeal cancers to genital warts (1). Their life cycle is inextricably linked to epithelial differentiation (2), and there are three phases of DNA replication during the viral life cycle (3). The viral replication factors E1 and E2 are expressed, and after the initial infection, the genome copy number increases to 20 to 50 per cell; this replication phase of the viral life cycle is called establishment. The proliferating infected cell begins to move up through the differentiating epithelium, and during this maintenance replication phase the viral genome copy number per cell May 2017 Volume 91 Issue 10 e00102-17

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remains steady at 20 to 50 copies. Following differentiation and the cessation of host cell replication, the viral genome undertakes a third type of replication in the upper layer of the epithelium, amplification, where the viral genome copy number increases to around 1,000 per cell. Our understanding of the host proteins that regulate the three different replication phases remains incomplete. The E2 proteins of all human papillomaviruses form homodimers via a carboxylterminal domain that then recognizes and binds to four 12-bp palindromic sequences in the long control region (LCR) (4); the LCR is a region that regulates transcription from the viral genome and also contains the viral origin of replication (5). Three of the E2 binding sites surround the A/T-rich origin of replication, and following binding to its target sequences the amino-terminal domain of E2 recruits the viral helicase E1 to the origin of replication via a protein-protein interaction. E1 then forms a dihexameric structure that binds to DNA polymerases and executes viral DNA replication. To enhance understanding of the cellular partners involved in E1-E2 replication, we identified novel partners for the HPV16 E2 protein amino-terminal domain, and one of the proteins identified was TopBP1 (6). TopBP1 has 9 BRCT (BRCA1 carboxyl-terminal) domains that act as hydrophobic interacting pockets for phosphopeptides, proteins, and damaged DNA structures (7). TopBP1 is involved in sensing and signaling the DNA damage response, interacts with Treslin during G1-S transition to activate replication initiation, and regulates the transcriptional activity of cellular proteins such as p53 and E2F1 (8, 9). This central role of TopBP1 in nucleic acid metabolism, particularly the role in initiation of DNA replication, made it an attractive candidate for mediating HPV16 DNA replication via interaction with E2. A mutant of E2 that had compromised interaction with TopBP1 was generated, and this mutant had reduced DNA replication function in association with E1 (10, 11). When introduced into the full HPV16 genome, the mutant failed to establish episomes following immortalization of human foreskin keratinocytes. The results demonstrate that there is an important role for TopBP1 in the HPV16 life cycle via interaction with E2. The role of TopBP1 in nucleic acid metabolism makes it an essential protein, so disrupting this protein to target viral replication is not a good strategy for combating HPV infections. Therefore, we searched for cellular enzymes that functionally interact with TopBP1; one such enzyme is the class III histone deacetylase SIRT1 (12). In response to glucose starvation, SIRT1 is activated in order to promote glycogen breakdown and gluconeogenesis (12). Following DNA damage, SIRT1 targeting of p53 is inhibited, resulting in acetylation followed by stabilization and execution of the p53 response (13). Recent publications demonstrate that (i) following glucose starvation, activated SIRT1 deacetylates TopBP1, resulting in disruption of the interaction with Treslin and preventing replication initiation, and (ii) DNA damage blocks SIRT1 action on TopBP1, resulting in increased acetylation, promoting the interaction of TopBP1 with Rad9 that subsequently results in activation of the ATR pathway and cell cycle arrest (14, 15). These papers were the first to demonstrate a functional SIRT1TopBP1 axis that responds to metabolic and DNA damage stress. Short hairpin RNA (shRNA) knockdown of SIRT1 reduces maintenance of HPV31 genomes and blocks viral amplification following differentiation in cervical keratinocytes (16). The reduction of SIRT1 affected transcription from the viral genome and blocked recruitment of the DNA damage and repair factors Nbs1 and Rad51 to the viral genome. Such recruitment is required for amplification of the viral genome during differentiation (17–20), and failure to recruit these factors could block amplification. However, a precise mechanism for SIRT1 in regulation of HPV transcription and replication is not known. The association with TopBP1 and the key role for SIRT1 in the HPV31 viral life cycle prompted us to investigate whether SIRT1 regulates HPV16 E1-E2 replication directly and, if so, how. The results demonstrate that SIRT1 is in the same cellular complex as E1 and E2 in C33a cells, and that it is recruited to the viral origin of replication in an E1-E2-dependent manner. Deletion of the SIRT1 gene using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology in C33a cells results in elevated levels of E1-E2 replication that are, at least in part, regulated by increased acetylation and stability of May 2017 Volume 91 Issue 10 e00102-17

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FIG 1 SIRT1 is a member of the E1-E2 replication complex in C33a cells. Cells were mock transfected (lane 1) or were transfected with E1 alone (lane 2), E2 alone (lane 3), or E1⫹E2⫹pOri (a HPV16 origin of replication-containing plasmid) (lane 4). Forty-eight hours following transfection, protein extracts were harvested from the cells. (A) Western blot showing the expression levels of endogenous SIRT1 and TopBP1 and the levels of E1 and E2 expressed from the transfected plasmids. ␤-Actin is shown as a loading control. (B) The protein extracts were immunoprecipitated with an SIRT1 antibody and the precipitates resolved using SDS-PAGE and Western blotting carried out for SIRT1, TopBP1, E1, and E2.

the E2 protein. Overall, the results demonstrate that SIRT1 is a functional member of the E1-E2 replication complex. Activating and/or inhibiting the enzymatic role of SIRT1 in HPV16 replication is a novel therapeutic opportunity for directly disrupting the viral life cycle. RESULTS SIRT1 is a member of the E1-E2 replication complex in vivo. As SIRT1 is a partner protein for TopBP1 and is required for the HPV31 life cycle, the ability of SIRT1 to complex with E1 and E2 was investigated. C33a cells were transfected with expression plasmids for E1, E2, or E1-E2 plus an HPV16 origin-containing plasmid, pOri. Proteins were harvested from the transfected cells 48 h later and the expression of the viral proteins confirmed (Fig. 1A). A SIRT1 antibody then was used to immunoprecipitate (IP) this extract, and the IP was blotted for SIRT1, TopBP1, E1, and E2 (Fig. 1B). From Fig. 1B it is clear that SIRT1 complexes with E1 (lane 2), E2 (lane 3), and both of them together (lane 4), as the SIRT1 antibody coimmunoprecipitates the viral proteins. The coimmunoprecipitation of TopBP1 by SIRT1 confirms the work of others and validates our approach of investigating TopBP1 interactors as being in complex with E1 and E2. These results do not confirm whether the binding of SIRT1 with the replication complex is direct or indirect. The ability of SIRT1 to be recruited to the viral origin of replication by E1-E2 then was tested (Fig. 2). C33a cells were transfected with pOri only (a plasmid containing the HPV16 origin of replication) (lane 1), pOri⫹E1 (lane 2), pOri⫹E2 (lane 3), or pOri⫹E1⫹E2 (lanes 4 and 5). Forty-eight hours later, chromatin was prepared from the transfected cells and chromatin immunoprecipitation (ChIP) was carried out. As there will be more DNA present following E1-E2 replication, we normalized all signals to the input pOri present in the chromatin as described in Materials and Methods. The results were then expressed relative to the levels obtained with immunoprecipitated pOri⫹E1⫹E2 and the specific antibodies equaling 1. As demonstrated previously, E1 and E2 are recruited to the viral origin of replication only when both viral proteins are expressed (Fig. 2A and B, lanes 4). Either protein by itself is present at very low levels. Significantly, SIRT1 is recruited to the origin of replication in an E1-E2-dependent manner (Fig. 2C, lane 4). This is identical to both Brd4 and TopBP1 (11). ChIP assay with the samples used in lane 4 was also carried out with rabbit serum (Fig. 2A to C, lane 5) and no signal was detected, demonstrating the signal detected in lane 4 is specific for the antibodies tested. May 2017 Volume 91 Issue 10 e00102-17

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FIG 2 SIRT1 is recruited to the HPV16 origin of DNA replication in an E1-E2-dependent manner. C33a cells were transfected with pOri (lane 1), pOri⫹E1 (lane 2), pOri⫹E2 (lane 3), or pOri⫹E1⫹E2 (lanes 4 and 5). Chromatin was prepared from the transfected cells after 2 days and immunoprecipitated with specific antibodies (lanes 1 to 4) against HA (that targets E1) (A), E2 (B), and SIRT1 (C). In lanes 5, immunoprecipitation was carried out with a rabbit serum as a nonspecific control. Following immunoprecipitation, DNA was rescued and quantified using real-time PCR targeting pOri. The input DNA in each sample was also determined using real-time PCR. The signal in each sample was then determined relative to the amount of pOri present in the input chromatin. The results are presented as the signal obtained, with the value for pOri⫹E1⫹E2 equaling 1. The results presented represent the summary of three independent experiments. Statistics demonstrated that the signal obtained in lane 4 in all samples was significantly greater than those in lanes 1 to 3 and 5 in all samples, with P values less than 0.05.

Generation of CRISPR/Cas9 SIRT1 knockout cell lines from C33a. Using a SIRT1 double nickase plasmid, the SIRT1 gene was targeted by the CRISPR/Cas9 system in C33a cells and potentially targeted clones selected using puromycin. Individual clones were isolated using cloning rings, trypsinized, transferred into 24-well plates, and subsequently expanded into cell lines. A pool of targeted cells was also generated by trypsinizing the remaining clones together and expanding them into a cell line. Following expansion, Western blotting was carried out to identify those clones that had been successfully targeted. Figure 3A demonstrates that SIRT1 knockout clones were generated; lane 1 is the parental C33a line, lanes 2 to 5 are individual clones, and lane 6 is a pool of clones that had been targeted for SIRT1 disruption. In lane 2 it is clear that clone 1 has almost no detectable SIRT1 expression, while in lane 6 the pool of cells has severely attenuated SIRT1 expression. The DNA region targeted by CRISPR/Cas9 was amplified in clone 1 by PCR and sequenced to confirm disruption of the SIRT1 gene (Fig. 3C). For subsequent experiments, clone 1 cells and pooled cells were used side by side to confirm that any effects observed were not clonally specific. In Fig. 3B it is demonstrated that both wild-type (WT) SIRT (pFlag-SIRT1-WT) (lanes 2, 5, and 8) and a deacetylase mutant (H363Y; pFlag-SIRT1-MT) (lanes 3, 6, and 9), which were Flag tagged (obtained from Addgene), could be expressed in wild-type C33a (lanes 1 to 3) cells as May 2017 Volume 91 Issue 10 e00102-17

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FIG 3 Generation of SIRT1 knockout clones using CRISPR/Cas9. (A) C33a cells were transfected with a SIRT1 double nickase plasmid and selected with 1 ␮g/ml puromycin for 2 weeks. Clonal cell lines were isolated and expanded and the remaining clones trypsinized to generate a pool. Extracts were prepared and analyzed by Western blotting for SIRT1 (upper) and ␤-actin (lower) expression. (B) Lanes 1 to 3 represent C33a cells, lanes 4 to 6 represent clone 1 (lane 2 in panel A), and lanes 7 to 9 represent the pooled line (lane 6 in panel A). Cells were mock transfected (lanes 1, 4, and 7), transfected with a Flag-tagged wild-type SIRT1 expression plasmid (lanes 2, 5, and 8) or a Flag-tagged deacetylase mutant SIRT1 (lanes 3, 6, and 9), and subjected to Western blotting for endogenous SIRT1 (upper), Flag-tagged SIRT1 (middle), and ␤-actin (lower). (C) The segment of DNA from clone 1 that was targeted by the nickase plasmid was PCR amplified and sequenced, and deletion of the targeted sequence (indicated by dashes) was confirmed.

well as in clone 1 (lanes 4 to 6) and pooled (lanes 7 to 9) cells. In Fig. 3B, the upper gel shows both endogenous and overexpressed SIRT1, the middle gel shows Flag-tagged SIRT1 proteins, and the bottom gel is a ␤-actin loading control. The SIRT1 knockout cells grew slower, and this was confirmed in a growth curve (Fig. 4A). The parental C33a cells grow significantly quicker than clone 1 and pooled cells, with pooled cells being somewhat intermediate in their growth rate, as might be expected for a mixed population of cells. Sirt1 knockout mouse cells have an increased frequency of replication origin firing during S phase, and we predicted that the clone 1 and pooled cells would have an extended S phase. To investigate this further flow cytometry was carried out, and the results are shown in Fig. 4B and summarized in Fig. 4C. In Fig. 4C it is clear that in both clone 1 and pooled cells there is an increase in the number of S phase cells. This was repeated with essentially identical results. Therefore, we propose that the reason the C33a cells grow faster than clone 1 and pooled cells is because of an extended S phase in the latter two cell lines. Deletion of SIRT1 boosts HPV16 E1-E2-mediated DNA replication. Transient DNA replication assays were carried out in C33a, clone 1, and pooled cells by transfecting them with pOri along with expression vectors for HPV16 E1 and E2. Lowmolecular-weight DNA was harvested from the transfected cells and treated with DpnI (that cuts only the input bacterially produced DNA) and exonuclease III (which degrades the DpnI-digested fragments) and the remaining pOri monitored using real-time PCR. This technique was first developed in our laboratory (21, 22). The results are shown in Fig. 5. The control lanes with pOri by itself or with E1 or E2 are not shown for simplicity, as the signal generated is several orders of magnitude below that detected with pOri with E1 and E2. All results are set to the levels of pOri⫹E1⫹E2 in C33a cells equaling 1 (lane 1). To confirm that all cell lines were transfected equally, input DNA levels from the control samples were determined and the results are shown in Fig. 5B; there is minimal variability in transfection efficiency between the cell lines. This is accomplished by digesting the samples with MboI, which recognizes only the freshly replicated DNA, and treating them with exonuclease III to remove any residual DNA fragments, followed by PCR detection of the input, pOri. As is clear in Fig. 5B, there is May 2017 Volume 91 Issue 10 e00102-17

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FIG 4 Cells without SIRT1 grow slower and have an extended S phase compared with parental C33a cells. (A) A growth curve was carried out over 6 days and the accumulated number of cells plotted on a log graph. The standard errors are so low that they do not show on this log-scale figure. (B) Flow cytometry was carried out to investigate the cell cycle profile of C33a, clone 1, and pooled cells. The sub-G1, G1, S, and G2/M cell populations are highlighted. This is a representation from one experiment that has been repeated at least one other time for these lines. (C) A graphic representation of the cell cycle profile of the C33a, clone 1, and pooled cells shown in panel B. The flow cytometry experiment was repeated and essentially identical results were obtained.

no significant difference in the transfected DNA in any of the samples, demonstrating that all cells are able to receive DNA to a similar degree. In the absence of SIRT1, E1-E2 replication increases, as shown for clone 1 and pooled cells (Fig. 5A, compare lanes 4 and 7 with lane 1). Transfection of pFlag-SIRT1-WT or pFlag-SIRT1-MT had no effect on E1-E2 replication in C33a cells (Fig. 5A, compare lane 1 with 2 and 3). However, in clone 1 and pooled cells, both SIRT1 expression vectors restored replication levels to those of C33a cells (lane 1). While the repression of replication by pFlag-SIRT1-MT was not quite as strong as that of pFlag-SIRT1-WT, there were no statistically significant differences between the proteins. Statistical analysis demonstrated significant differences between lanes 1 and 4 and also between lane 4 and lanes 5 and 6, which is indicated by the brackets and associated P values. The conclusions from these experiments are that SIRT is in the E1-E2 replication complex and its removal elevates DNA replication. This elevation can be restored to wild-type levels by both wild-type and deacetylase mutant SIRT1 expression vectors. SIRT1 controls the acetylation status of E1 and E2. One explanation for the elevated E1-E2 replication in the absence of SIRT1 is an increased level of the replication factors. Figure 6A demonstrates that E2 (lanes 1 and 2) or E1 (lanes 3 and 4) protein levels are not dramatically affected by the absence of SIRT1. However, when E1 and E2 are coexpressed either without (lanes 5 and 6) or with (lanes 7 and 8) pOri, it is clear that the absence of SIRT1 elevates the expression of the replication factors (compare lane 6 with 5 and lane 8 with 7), with E2 being particularly affected. To investigate whether this was due to the enzymatic activity of SIRT1, clone 1 cells were transfected with pOri⫹E1⫹E2 and cotransfected with control plasmid, pFlag-SIRT1-WT, or pFlagMay 2017 Volume 91 Issue 10 e00102-17

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FIG 5 Absence of SIRT1 enhances E1-E2-mediated DNA replication. E1-E2-mediated DNA replication assays were carried out in C33a (lanes 1 to 3), clone 1 (lanes 4 to 6), and pooled cells (lanes 7 to 9) cells. Wild-type SIRT1 (lanes 2, 5, and 8) and mutant (MT) SIRT1 (lanes 3, 6, and 9) were cotransfected into the assay. (A) DNA replication signal, obtained as measured following DpnI and exonuclease III treatment of harvested DNA that degrades the transfected input plasmids, followed by real-time PCR to detect freshly replicated pOri. (B) The same samples following Mbo1 and exonuclease III treatment of harvested DNA, which degrades the replicated DNA, followed by real-time PCR to detect input pOri. Results represent a summary of at least 3 independent experiments, and standard error bars are shown. Significant differences in the levels of samples shown in panel A are represented by the brackets, and associated P values are shown.

SIRT1-MT. Proteins were harvested 48 h later and Western blotting was carried out, and the results are shown in Fig. 6B. pFlag-SIRT1-WT was able to significantly reduce the levels of E2 protein with a slight reduction of E1 (compare lane 6 with 7). pFlagSIRT1-MT reduced E2 levels a little (compare lane 6 with 8) and had no effect on E1 levels. This experiment was repeated and the blots quantified, and the results are presented in histogram format (Fig. 6C). The quantitation demonstrates that wild-type SIRT1 can reduce E2 levels significantly and that the mutant SIRT1 also does this but to a significantly lesser degree. To confirm that any effect SIRT1 had on the expression levels of the viral replication factors was not related to alteration of RNA levels in the SIRT1 knockout cells, the RNA levels of E1 and E2 were measured. RNA was harvested and converted to cDNA, and real-time PCR detecting E2 (Fig. 6D) and E1 (Fig. 6E) was carried out. Figure 6D and E represents the summary of three independent experiments demonstrating that there is no significant alteration of E1 and E2 RNA levels in the SIRT1 knockout cells. The results shown in Fig. 6 suggest that the acetylation status of the viral proteins was altered in the absence of SIRT1. To investigate this possibility, an immunoprecipiMay 2017 Volume 91 Issue 10 e00102-17

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FIG 6 Lack of SIRT1 increases E2 protein levels. (A) C33a (lanes 1, 3, 5, and 7) or clone 1 (lanes 2, 4, 6, and 8) cells were transfected with the indicated plasmids, and protein extracts were prepared 48 h later. Western blot analyses then were carried out with the indicated antibodies. (B) C33a (lanes 1 and 2) or clone 1 (lanes 3 to 8) cells were transfected with the indicated plasmids, and protein extracts were prepared 48 h later. Western blots then were carried out with the indicated antibodies. The experiment was repeated and the levels of proteins quantified as described in Materials and Methods. (C) The results of this quantitation for lanes 6 to 8 in panel B are represented graphically and represent the summary of two independent experiments. Statistically significant differences in the protein levels are indicated. To confirm that the alterations in E1 and E2 protein levels were not due to alterations in RNA levels, E2 (D) and E1 (E) RNA levels were determined

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FIG 7 SIRT1 regulates the acetylation status of E2 and E1. (A) The protein extracts used for Fig. 6B were immunoprecipitated with an antibody that recognized acetylated lysine residues and subjected to Western blotting with an HA antibody (that detects E1) and an E2 antibody. The faint band in lane 1 is antibody background that was detected very occasionally, and there is no E2 in this lane. (B) The bands for E2 were quantitated for lanes 6 to 8. The change of E2 acetylation in the presence of SIRT1 was reproducible and observed in additional experiments.

tation with an acetylated-lysine antibody and Western blotting were carried out. The results from this experiment are shown in Fig. 7; the samples used for this experiment were those used in the experiments shown in Fig. 6B, which can be used as a measure of input into the coimmunoprecipitation. In wild-type C33a cells there is very little acetylation of either E1 or E1 (lane 2). In the absence of SIRT1 there is increased acetylation of both E1 and E2 by themselves (lanes 4 and 5, respectively). When both are present in clone 1 cells, there is a clearly increased acetylation of both E1 and E2 compared with C33a cells (compare lane 6 with 2). The addition of pFlag-SIRT1-WT decreased the acetylation of the proteins, while pFlag-SIRT1-MT had little effect. A faint background band was seen in lane 1 which was nonspecific, as there is no E2 present in this sample. Lanes 6 to 8 were quantitated and the results are shown in Fig. 7B. The conclusion from these experiments is that SIRT1 targets E1 and E2 for deacetylation.

FIG 6 Legend (Continued) using real-time PCR analysis of cDNA prepared from C33a (lanes 1 and 3) and clone 1 (lanes 2 and 4) cells. The results are expressed relative to the GAPDH levels in each sample. The results shown in panels D and E represent the summary of three independent experiments, with standard error bars shown. There is no significant difference in the E2 and E1 RNA levels between wild-type C33a and clone 1 cells. May 2017 Volume 91 Issue 10 e00102-17

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FIG 8 Acetylated E2 is stabilized in the absence of SIRT1. (A and B) C33a (top) and clone 1 (bottom) cells were mock transfected (lane 1) or transfected with E1⫹E2⫹pOri (lanes 2 to 7). Prior to protein harvest 48 h following transfection, cycloheximide was added to the cells for the times indicated. Western blotting for E1, E2, and ␤-actin then was carried out. (C) The experiment described for panel B was repeated and the Western blots quantitated, and the results are shown. (D) The extracts from C33a and clone 1 cells Western blotted in panels A and B were immunoprecipitated with an antibody binding acetyl lysine residues, and Western blotting for E2 was carried out.

The next experiment tested whether the increased acetylation of the viral proteins is responsible for the elevated levels of the viral proteins. Acetylation of E2 stabilizes the protein. C33a and clone 1 cells were transfected with E1⫹E2⫹pOri plasmids and treated with cycloheximide for the times indicated prior to harvest. All cells were harvested after 48 h. Figure 8A demonstrates that in C33a cells E1 is stable while E2 is not, as we have demonstrated before (23). However, in clone 1 cells (Fig. 8B) there is an increased stability of the E2 protein, although we noted a slight reduction in E2 at earlier time points in the absence of SIRT1 (Fig. 8B, middle, compare lane 2 with 3). This experiment was repeated, and the blots were quantified and plotted in histogram format (Fig. 8C). The C33a and clone 1 cycloheximide-treated extracts shown in Fig. 8A and B were used for acetyl lysine IP followed by blotting for E2 (Fig. 8D). In C33a cells there is an acetylated band in the untreated cells that disappears 1 h following cycloheximide treatment. However, for clone 1 cells there is a level of acetylated E2 protein that is present in untreated cells and remains unchanged following 5 h of cycloheximide treatment. This demonstrates that the acetylated E2 protein is remarkably stable in the absence of SIRT1, while the acetylated E2 observed in the wild-type C33a cells is unstable. Therefore, the consequence of acetylation of E2 changes dramatically in the absence of SIRT1. May 2017 Volume 91 Issue 10 e00102-17

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DISCUSSION The results presented here demonstrate that SIRT1 is part of the HPV16 E1-E2 DNA replication complex and is recruited to the viral origin of DNA replication in an E1-E2-dependent manner. The results presented do not determine whether there is a direct interaction between the viral proteins and SIRT1 or whether there is a bridge protein, such as TopBP1, that brings the complex together. The absence of SIRT1 elevates replication by the viral proteins, and this could be mediated by several mechanisms. First, elimination of SIRT1 extends the S phase, and this could allow for an increase in E1-E2 replication, which is not controlled in a cell cycledependent manner, and the longer the S phase the greater the opportunity for E1-E2 replication to occur. We do not favor this mechanism, as the increase in replication is around 4-fold in the absence of SIRT1 and the increase in S phase cells is only in the region of 30%. A second possibility is that the increased stability of E2 via acetylation in the absence of SIRT1 is responsible for the enhanced replication. We have shown previously that titration of E2 into these DNA replication assays can regulate the level of replication (21). Other mechanisms may also exist, including alteration of chromatin modifications surrounding the viral origin, but the increased levels of E2 protein in the absence of SIRT1 would explain the enhanced DNA replication. To confirm that the increased E2 acetylation and DNA replication is due to the absence of SIRT1, we coexpressed both wild-type SIRT1 and a deacetylase mutant SIRT1, along with E1-E2 in replication assays and biochemical analysis of the complex. In the biochemical assays, the wild-type SIRT1 reduced E2 protein levels in the clone 1 cells (Fig. 6B and C), while the deacetylase mutant did so to a lesser extent. However, in DNA replication assays both the wild-type and mutant SIRT1 restored E1-E2 replication to the level in wild-type C33a cells when coexpressed in the clone 1 cells (Fig. 5). This difference could be due to the level of E2 protein that is used in both experiments. In order to obtain a nonsaturated level of DNA replication in the DNA replication assays, only 10 ng of E2 DNA is used in the assays, while for the biochemical assays 2,000 ng is used in order to be able to detect the E2 protein in Western blots. The lower level of E2 in the replication assays therefore could be targeted by residual deacetylase activity retained in the mutant SIRT1 protein, while in the biochemical analysis there is too much E2 protein for the compromised mutant to deacetylate E2. However, it is also entirely possible that the SIRT1 protein has a dual role in replication that is both enzymatic and structural. An investigation of the role of SIRT1 in the HPV31 life cycle concluded that there was also a structural role for SIRT1 in the viral life cycle (16), suggesting that both the deacetylase function of SIRT1 along with a structural component regulate the HPV31 life cycle. A previous study observed that SIRT1 is important for the HPV life cycle, in agreement with other studies (16). Langsfeld and colleagues demonstrated, using shRNA targeting SIRT1, that SIRT1 is required for the maintenance stage of the viral life cycle. They showed that SIRT1 depletion is important for transcriptional control of the viral genome during differentiation. They also demonstrated that SIRT1 depletion alters the chromatin modification of the viral genome, influencing the recruitment of DNA repair factors that are thought to be required for viral genome amplification. Our current report adds another function for SIRT1 in regulation of the viral life cycle: regulation of E1-E2-mediated DNA replication. In the DNA replication assays carried out in C33a cells, the amplification stage of the viral life cycle is modeled; viral replication factor levels are high along with DNA replication levels, and large nuclear foci are formed that are observed only in differentiated cells containing HPV31 where viral genome amplification occurs (24). It is also possible that there is a fundamentally different role for SIRT1 in the life cycle of HPV31 and the life cycle of HPV16. Recently we demonstrated that this is the case for Brd4 interaction with E2, which is essential for the HPV16 life cycle and E1-E2 DNA replication (11), while others have demonstrated this is not essential for the May 2017 Volume 91 Issue 10 e00102-17

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life cycle of HPV31 (25). Therefore, much work is required to fully understand the role of SIRT1 in both the HPV31 and HPV16 life cycles. Another role for SIRT1 in HPV16 E1-E2-mediated DNA replication could be related to regulation of the quality of replication. E1-E2 replication activates the DNA damage response (DDR) in C33a cells (26), and this is not altered in clone 1 cells (not shown), which is in agreement with SIRT1 not being required for activation of the DDR (27, 28). The activation of the DDR stimulates SIRT1 to deacetylate Nbs1 (28), and this deacetylation makes Nbs1 a substrate for the ATM kinase (29, 30). Phosphorylation of Nbs1 then promotes the formation of the MRN complex that is recruited to sites of DNA damage in order to promote homologous recombination via subsequent recruitment of cellular repair factors, such as Rad51. HPV use a very similar mechanism. They require activation of the DDR for the amplification phase of the viral life cycle, and it has been shown that the MRN complex and other DDR factors are recruited directly to the viral genome (17–20). Nbs1 is required for the viral life cycle (20). One hypothesis is that this recruitment results in the virus using homologous recombination in order to amplify its genome in the upper layer of the infected epithelium (31). In clone 1 cells, the initial DDR activated by E1-E2 replication in C33a cells is not abrogated, but the downstream consequences of this signaling could very well be disrupted. The role of SIRT1 in regulation of mammalian genomic stability is well established (32–34). We hypothesize that even though there are elevated levels of E1-E2 DNA replication in the absence of SIRT1, this replication could be of poor quality. We have recently demonstrated that the treatment of cells with etoposide does not arrest E1-E2 replication but does reduce the quality of replication, and an identical observation can be made following the elimination of TopBP1 from the cell (26, 35). Another important role for SIRT1 in regulation of the HPV16 life cycle could be the regulation of viral genome amplification in the upper layers of the differentiated epithelium. In this part of the infected epithelium, the E2 protein is stabilized by an unknown mechanism (36) and the viral genome is amplified from around 20 to 50 copies per cell to around 1,000; this amplification is at least in part mediated by the stabilization of the E2 protein. It is intriguing that in the paper on the HPV31 life cycle, in differentiated cells SIRT1 is no longer associated with the viral genome, even though it is expressed in the differentiated cells (16). Therefore, the absence of SIRT1 from the viral genome could result in acetylation of E2 as we describe in this report; others have demonstrated that acetylation can regulate E2 function (37). Tip60 is a candidate for mediating this acetylation. It is required for the HPV31 life cycle (38), and our preliminary studies demonstrate that Tip60 is recruited to the viral origin of replication in an E1-E2-dependent manner (data not shown). It also should be noted that SIRT1 can regulate the function of Tip60: deacetylation represses Tip60 acetylase function (39– 41). Therefore, we hypothesize that SIRT1 controls the levels of viral DNA replication via deacetylation of E2 and that in differentiated cells the disconnection of SIRT1 from the viral genome allows a cellular acetylase, such as Tip60, to acetylate and stabilize E2 and therefore promote the viral life cycle. In this report, we have demonstrated an essential role for SIRT1 in regulating the DNA replication properties of E1 and E2 in C33a cells via regulation of the acetylation status of the viral proteins. Our acetylation studies also demonstrate that the absence of SIRT1 alters the posttranslational modifications on E2 and the stability of the protein. In the presence of SIRT1 acetylated E2 is unstable (Fig. 8), while in the absence of SIRT1 acetylated E2 is stable. It is also of note that in the absence of SIRT1 the nonacetylated E2 protein is extremely unstable, more so than in the wild-type C33a cells (Fig. 8). Therefore, the absence of SIRT1 is having a pleiotropic effect on the posttranslation modification of E2. There are several possible roles that SIRT1 plays in the viral life cycle via this regulation, such as promotion of homologous recombination for amplification of the viral genome and stabilization of E2 in differentiated epithelium that would promote viral genome amplification. Future studies will focus on enhancing our understanding of the role May 2017 Volume 91 Issue 10 e00102-17

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of SIRT1 in the viral life cycle and determining whether therapeutic targeting of SIRT1 could alleviate HPV infection and disease. MATERIALS AND METHODS Cell line, plasmids, and reagents. C33A cells (ATCC HTB-31) were grown in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified CO2 incubator in 5% CO2 at 37°C. Details of the HPV16-E2 and hemagglutinin-E1 (HA-E1) expression plasmid were provided previously (42, 43). For the replication assay, pOri plasmid was used as described previously (21, 22). For the SIRT1 knockout using CRISPR, SIRT1 double nickase plasmid (sc-400085-NIC) was purchased from Santa Cruz. After transfection, cells were selected using puromycin. FLAG-WT-SIRT1 (no. 1791) and FLAG-MTSIRT1 (H363Y) (no. 1792) plasmids were purchased from Addgene. SIRT1 antibody (07–131) and acetyl lysine antibody (AB3879) were obtained from EMD Millipore. Analysis of cell cycle profile by propidium iodide staining and flow cytometry. Cells were washed with ice-cold phosphate-buffered saline (PBS) supplemented with 1% FBS. The cells were then resuspended in 3 ml of chilled 70% ethanol and kept at 4°C for at least 30 min. Cells were pelleted by centrifuging at 500 ⫻ g for 5 min and washed in ice-cold PBS. The pellets were suspended in 500 ␮l PBS containing 50 g/ml propidium iodide and 100 g/ml RNase A, incubated at 37°C for 30 min, and then analyzed using a Guava EasyCyte mini flow cytometer (Guava Technologies, USA). Replication assay. For the replication assay, cells were plated at a concentration of 5 ⫻ 105 in a 100-mm tissue culture disc. After 24 h the cells were transfected with 10 ng pOriM, 1 ␮g E1, and 10 ng E2 plasmids using calcium phosphate. The following day, the cells were washed twice with PBS and replenished with fresh medium. Forty-eight hours posttransfection, the cells were harvested using Hirt solution (10 mM EDTA, 0.5% SDS) and the samples were processed for quantitative PCR (21, 22). Western blotting. Cells were harvested using 0.05% trypsin and were pelleted by centrifugation. The pellets were washed twice with ice-cold PBS and then resuspended in lysis buffer (0.5% Nonidet P-40 [NP-40], 50 mM Tris, pH 7.8, 150 mM NaCl with protease and phosphatase inhibitor cocktail). The cells were lysed and the proteins were obtained as described previously (11). Protein concentration was determined by bicinchoninic acid (BCA) assay (Sigma) using bovine serum albumin (BSA) as a standard. Approximately 50 ␮g protein was run on a 4 to 12% gradient gel (Invitrogen) and transferred onto a nitrocellulose membrane. Odyssey blocking buffer was used to block the membranes as well as for antibody dilution. After blocking, the membranes were incubated with primary antibodies. PBS-Tween solution (0.1%) was used to wash the membranes, after which they were incubated with Odyssey secondary antibodies. The membranes were washed again with 0.1% PBS-Tween solution before imaging using the Odyssey Li-Cor imaging system. The images were quantified using Image Studio Lite software, version 5.2. IP. A total of 200 ␮g of protein lysate was taken, and the volume was increased up to 300 ␮l using the lysis buffer. Two micrograms of antibody was added to each lysate, which were rotated overnight on a rotor at 4°C. The following day, a protein A-Sepharose bead slurry was washed five times with lysis buffer and then equilibrated with the same lysis buffer so that equal volumes can be added to each of the lysates. The lysate-bead mixture was rotated on a rotor at 4°C for 5 h, after which they were again washed five times with the lysis buffer to remove nonspecific binding. The beads were then processed for Western blotting. ChIP. For ChIP, cells were plated at a concentration of 6 ⫻ 105 in a 100-mm2 dish. The following day cells were transfected with 1 ␮g of pOriM, 1 ␮g of E1, and 2 ␮g of E2 plasmid using the CaPO4 precipitation method. The next day the cells were washed with PBS and transferred to 15-cm2 dishes. Forty-eight hours posttransfection the cells were harvested and processed for chromatin as described previously (11). The chromatin concentration of each sample was determined using a NanoDrop spectrophotometer. One hundred micrograms of chromatin was used for each experiment. Two micrograms of desired primary antibodies and 20 ␮l of A/G magnetic beads were used for each sample. Chromatin pulldown and the washing of beads was carried out; after washing of beads, the chromatin was processed for quantitative PCR (qPCR) (11). TaqMan qPCR using the pOriM primer and probe set (21, 22) was used to investigate the presence of each protein at the HPV16 origin of replication. To measure the amount of pOriM DNA in each sample, 100 ␮g of chromatin was processed to rescue the DNA, and the pOriM quantity was measured by qPCR relative to values for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The values were then normalized from the antibody pulldown DNA values to the input to omit the variation of unequal pOriM levels in different samples. Rabbit serum was used for the negative control. The results are expressed relative to the pOri⫹E1⫹E2 samples, the value of which equaled 1. Growth assay. Cells were plated at a concentration of 1 ⫻ 106 per plate. The cells were allowed to grow for 2 days and then were trypsinized and counted. A total of 1 ⫻ 106 cells from each sample again were used for plating and allowed to grow for another 2 days. The procedure was repeated for another 2 days. After 6 days in total, the cell counts were summed and plotted on a graph in a log scale using Microsoft Excel. Cycloheximide time chase. Forty-eight hours posttransfection, cycloheximide (100 ␮g/ml) was added to the plates for various times. The plates were then incubated for the specific time periods and harvested using trypsin, and then Western blotting was carried out. The blots were then quantified with respect to ␤-actin. RNA assay. Cells were harvested and the RNA was extracted using the SV total RNA isolation system (Promega). Later, cDNA was synthesized from the RNA using a high-capacity cDNA reverse transcription May 2017 Volume 91 Issue 10 e00102-17

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kit with RNase inhibitor (Invitrogen). The samples then were processed for qPCR using E1- and E2-specific primers, and the data were normalized to results for GAPDH. Statistical analysis. A two-tailed Student’s t test was employed, and a P value of ⬍0.05 was considered statistically significant.

ACKNOWLEDGMENTS We thank Renfeng Li for critical readings of the manuscript. This work was funded with a faculty start-up package to I.M.M. from the VCU Philips Institute for Oral Health Research and with NCI Designated Massey Cancer Center grant P30 CA016059.

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