The Deacetylase Sirtuin 1 Regulates Human Papillomavirus Replication by Modulating Histone Acetylation and Recruitment of DNA Damage Factors NBS1 and Rad51 to Viral Genomes.

Human papillomaviruses (HPV) regulate their differentiation-dependent life cycles by activating a number of cellular pathways, such as the DNA damage response, through control of post-translational protein modification. Sirtuin 1 (SIRT1) is a protein deacetylase that modulates the acetylation of a number of cellular substrates, resulting in activation of pathways controlling gene expression and DNA damage repair. Our studies indicate that SIRT1 levels are increased in cells containing episomes of high-risk HPV types through the combined action of the E6 and E7 oncoproteins. Knockdown of SIRT1 in these cells with shRNAs impairs viral activities including genome maintenance, amplification and late gene transcription, with minimal effects on cellular proliferation ability. Abrogation of amplification was also seen following treatment with the SIRT1 deacetylase inhibitor, EX-527. Importantly, SIRT1 binds multiple regions of the HPV genome in undifferentiated cells, but this association is lost upon of differentiation. SIRT1 regulates the acetylation of Histone H1 (Lys26) and H4 (Lys16) bound to HPV genomes and this may contribute to regulation of viral replication and gene expression. The differentiation-dependent replication of high-risk HPVs requires activation of factors in the Ataxia Telangiectasia Mutated (ATM) pathway and SIRT1 regulates the recruitment of both NBS1 and Rad51 to the viral genomes. These observations demonstrate that SIRT1 is a critical regulator of multiple aspects of the high-risk HPV life cycle

SIRT1 binds the HPV31 URR and controls histone acetylation.

<p><b>(A)</b> CIN612 cells were grown undifferentiated in monolayer culture or induced to differentiate in high-calcium media for 72 hours and ChIP-pPCR was performed for the HPV URR. Results shown are the average Ct IP/Ct IgG using isotype matched IgG control antibody. Ct values were normalized to HPV genome input copy number. Error bars are +/- SEM of the average of three PCR replicates between three independent experiments. Significance of differences as determined by Student’s t-test is shown as * = p<0.05 <b>(B)</b> Histones were acid extracted and electrophoresed on SDS-PAGE gel. Total histone 3 (H3) serves as a loading control. <b>(C)</b> Monolayer CIN612 cells were transduced for 72 hours and ChIP-qPCR for the HPV31 URR performed as in (A). Results shown are the average fold enrichment relative to isotype matched IgG control antibody. Ct values were normalized to HPV genome input copy number. Error bars are +/- SEM of the average of three PCR replicates between three independent experiments.</p

HPV31 E6 and E7 both increase levels of SIRT1 protein in primary keratinocytes.

<p><b>(A)</b> HFK cells retrovirally transduced with either empty control vector or pLXSN encoding HPV31 E6, E7, or E6 and E7 together were stably selected with G418 and analyzed by Western blot for SIRT1. p53 protein levels was used as a positive control to confirm that E6 and E7 constructs were active. <b>(B)</b> Three independent experiments as in (A) were analyzed by Licor Image Studio software. Results were normalized to GAPDH and are expressed as fold change over pLXSN empty vector control. Error bars represent +/- 1 SEM. Significance as determined by Student’s t-test is shown as * = p<0.05 <b>(C)</b> RT-PCR performed on RNA isolated from the same cells as in (A and B). Results were normalized to signal obtained using GAPDH primers and is expressed as fold change relative to pLXSN empty vector control. Error bars represent +/- 1 SEM of the averages of 3 PCR replicates from each of 3 independent experiments. <b>(D)</b> HFK cells were retrovirally transduced with pLXSN vector control, wild-type HPV31 E6, E7, or mutants described in the text. Protein lysates were tested by western blot. <b>(E)</b> Three independent experiments, as in (D), were analyzed using Licor Image Studio software. Results were normalized to GAPDH and expressed as fold change relative to pLXSN control. Error bars represent +/- 1 SEM.</p

Depletion of SIRT1 by shRNA inhibits viral DNA replication and amplification

<p>(A) CIN612 cells stably transduced with shGFP control or two shSIRT1 constructs were induced to differentiate in high-calcium media for up to 96 hours. Southern blot was performed on extracted DNA from cells harvested at indicated times. (B) Signal intensity was quantified using LICOR image analysis software. Error bars indicate +/- 1 SEM of the values between at least three independent experiments. Significance of differences between shRNA knockdown cells and shGFP cells was determined by Student’s T-test and is indicated as * = p<0.05, ** = p<0.005, and *** = p<0.0005. ns = no significant difference between mock transduced and shGFP cells. (C) Protein lysates were collected from cells in (A) and Western blot analysis performed to confirm knockdown. Cytokeratin 10 (K10) serves as a marker of differentiation. Blot is representative of three independent experiments. (D) CIN612 cells were transduced with shGFP control or shSIRT1 lentiviruses and episome levels examined by Southern blot at each passage thereafter. SIRT1, SIRT6, and GAPDH protein levels were analyzed by western blot of lysates from cells taken from the same population as for the Southern blot. (E) Sub-saturated x-ray films were scanned and signal intensity was quantified using LICOR image analysis software. Episomal signal intensity at each timepoint was normalized to CIN612 Passage 0 levels. Error bars indicate +/-1 SEM of the values between at least three independent experiments. Significance was measured by Student’s t-test and is indicated as * = p < 0.05 and ** = p < 0.005. Significance for shSIRT1-1 is shown above the line and shSIRT1-5 is shown below. (F) CIN612 cells were treated for with either 80μM EX-527 or the same volume of DMSO for 48 hours, then harvested or induced to differentiate for the timepoints indicated. Southern blot was performed on DNA extracted from treated and control cells. Results shown are representative of three independent experiments.</p

SIRT1 knockdown diminishes binding of DNA damage factors to the HPV URR.

<p><b>(A)</b> CIN612 cells were mock transduced or transiently transduced with shGFP control or shSIRT1 for 72 hours and then induced to differentiate in high-calcium media for 72 hours. Protein lysates were analyzed by western blot. Images were captured using a Licor imaging system. <b>(B)</b> Monolayer CIN612 cells were transduced for 72 hours and ChIP-qPCR for the HPV31 URR performed. Results shown are the average fold enrichment relative to isotype matched IgG control antibody. Ct values were normalized to HPV genome input copy number. Error bars are +/- SEM of the average of three PCR replicates between three independent experiments. Significance as determined by Student’s t-test p-values are given.</p

Depletion of SIRT1 by shRNAs inhibits HPV late gene transcription and expression.

<p><b>(A)</b> Northern blot of RNA isolated from CIN612 cells stably selected to express control shRNA and shSIRT1 constructs. <b>(B)</b> Sub-saturated X-ray films from (A) were scanned and analyzed by Licor signal intensity analysis and expressed as fold change of late transcripts E1^E4 and E5 relative to CIN612 undifferentiated control. Error bars are +/- 1 SEM. <b>(C)</b> HFKs and HFKs stably expressing HPV31 and either shGFP or shSIRT1 were grown in organotypic raft culture for 14 days. HE is hematoxylin/eosin stain. E1E4 is an HPV31 late gene product and is expressed in upper layers of infected epithelium. K10 is a differentiation marker.</p

HPV induces overexpression of SIRT1 throughout keratinocyte differentiation.

<p><b>(A)</b> Western blot analysis of SIRT1 in normal HFKs. HFKs were stably transfected with HPV 31 or 16 genomes, and CIN612 cells. <b>(B)</b> Western blot analysis of other SIRT family members in the same cells as in (A) <b>(C)</b> Western blot analysis of HFKs and CIN612 cells induced to differentiate using 1.5mM calcium chloride for up to 96 hours. Cytokeratin 10 (K10) is a marker of differentiation. <b>(D)</b> Protein blot signal intensity analysis was performed using Licor Image Studio software on results from at least three independent experiments. Error bars represent +/- 1 SEM. Significance as determined by Student’s t-test is shown as * = p<0.05, ** = p<0.005.</p