Cutaneous Papillomavirus E6 Proteins Must Interact with P300 and Block P53-mediated Apoptosis for Cellular Immortalization and Tumorigenesis

# these authors contributed equally to this work Running title: Binding and inhibition of p300 by E6-proteins of CRPV and HPV38 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Binding of the papillomavirus E6 protein to E6AP and induction of p53 degradation is a common feature of genital high-risk human papillomaviruses (HPV), but cutaneous HPV lack this capacity. Nevertheless several cutaneous HPV types like HPV38 are associated with tumor formation when combined with a genetic predisposition, immunosuppression or UV-exposure. In an animal model system, the cottontail rabbit papillomavirus (CRPV) rapidly induces skin cancer without additional cofactors and CRPVE6 and E7 immortalize rabbit keratinocytes in vitro. However, CRPVE6 neither interacts with E6AP, with p53 nor induces p53 degradation. In this study, we show that interaction of CRPV or HPV38E6 with the histone acetyltransferase p300 is crucial to inhibit the ability of p53 to induce apoptosis. Strikingly, E6 mutants deficient for p300 binding are incapable of preventing p53 acetylation, p53 dependent transcription and apoptosis induction. Moreover E6 mutants deficient for p300 binding can not contribute to HPV38-induced immortalization of human keratinocytes or CRPV-induced tumor formation. Our findings highlight changes in the p53 acetylation status mediated by the viral E6 protein as a crucial requirement in the ability of high-risk cutaneous papillomaviruses to immortalize primary keratinocytes and induce tumors. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.


Introduction
Although more than 100 different human papillomaviruses (HPVs) were characterized on the basis of sequence homologies, only a limited number was shown to be associated with cancer development so far.Thirteen so called high-risk types of the alpha genus HPVs (α-HPVs) play a critical role in the development of cervical cancer and have been implicated in other anogenital cancers and a subset of head and neck carcinoma (1,2).In addition β-HPVs have been linked to the development of non-melanoma skin cancers (NMSC), which represents the most common malignancy in Caucasians worldwide (3).Beta-papillomaviruses such as HPV5 and 8 were described to be associated with skin carcinogenesis in patients with the rare genetic disorder epidermodysplasia verruciformis (EV).EV patients develop warts, which progress in up to 60% of the individuals mainly into primary squamous cell carcinoma (4).Furthermore, up to 70% of long-term immunosuppressed patients develop skin cancer of which more than 80% contain HPV-DNA (5)(6)(7).A causative relationship between β-HPV infection and NMSC development in immunocompetent individuals is suggested by epidemiological data and by the fact that the associated risk factors age, UV-mediated local and transplantation-related immunosuppression point to an infectious agent most probably belonging to the ß2-group that includes HPV38.(8,9).
An excellent animal model to study papillomavirus induced skin cancer formation is the New Zealand White rabbit, where infections with cottontail rabbit papillomavirus (CRPV) causes the development of tumors within 3 to 6 weeks post-infection that progress within 6 to 12 month into invasive cancer without additional cofactors (10,11).Recently, we reported that the CRPV oncoproteins E6 and E7 are able to immortalize primary rabbit keratinocytes (12),

Animal model
Infections of New Zealand White rabbits with CRPV DNA were performed as previously described (10).Tumor numbers and maximum diameter of each tumor were determined over a period of 5 months.Biopsies were removed 3 months post infection, embedded in Tissue-Tek O.C.T. compound (Sakura Finetek), snap-frozen, and stored at -80°C or DNA was extracted using a BioRobot EZ1 workstation with the EZ1 DNA Tissue Kit (Qiagen).Viral DNA was amplified using primers CRPV444-463F and CRPV1124-1104R and sequenced.

Immunofluorescence microscopy and TUNEL staining
Cells were grown on MatTek glassbottom culture dishes (MatTek Corporation), fixed for 10minutes in 2% paraformaldehyde and incubated with HA antibody (sc-805, Santa Cruz) in PBS/3% BSA for 1hour at RT, washed and incubated with FITC-conjugated anti-rabbit IgG antibodies.Cells were counterstained with DAPI.For TUNEL staining cells were fixed 48 hours post transfection with 4% neutral-buffered formaldehyde for 5 minutes and permeabilized with 20 μg/ml proteinase K (Merck) for 5 minutes at room temperature.The terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling assay (TUNEL) was performed using the QIA39 FragEL in situ apoptosis detection kit (Merck).counted to determine the rate of apoptosis.
Blots were developed with SuperSignal West (Thermo Scientific) substrate and visualized by the Fluor-S Max MultiImager (Bio-Rad).Band intensities were quantified using the Quantity One quantification software package (version 4) (Bio-Rad).

P53 is not degraded by E6 proteins of cutaneous papillomaviruses
The addition of the proteasome inhibitor MG132 to CRPV and HPV38 E6/E7 immortalized keratinocytes has indicated that the respective E6 proteins do not induce p53 degradation (12,24).To further confirm these findings, HPV16, HPV38 or CRPV E6/E7 immortalized keratinocytes were treated with cycloheximide, an inhibitor of protein biosynthesis (Fig 1A).detectable p53 levels, whereas p53 was unaffected in CRPV or HPV38 E6/E7 immortalized cells.Consistent with this MG132 treatment only increased p53 in HPV16 E6/E7 cells, but not in HPV38 E6/E7 cells.These data clearly show that p53 is not degraded by CRPV or HPV38 E6 proteins.

CRPVE6 represses transcriptional activity of p53
To evaluate if rabbit p53 (rp53) is similar to human p53 (hp53) with regard to transcriptional activation, we expressed rp53 and hp53 in p53-deficient Saos-2 cells and tested for p21 induction.After 48 hours a 4-fold induction of p21 was observed by rp53 and hp53.P21induction of rp53 was strongly impaired by cotransfection of CRPVE6 (Fig. 1B) although it does neither interact with nor degrade p53 (12,20,21).

CRPVE6 interacts with p300
Immobilized E6 proteins fused to maltose-binding protein (MBP) were incubated with HEK293T cell extracts containing high amounts of p300 protein.Similar to high-risk HPV, CRPV encodes two forms of E6.Short E6 (SE6) is generated by the second methionine (M98) within the full length E6 (long E6, LE6).Both CRPVE6 proteins revealed a much stronger interaction with p300 than the E6 proteins of HPV11, 16 or 18 (Fig 1C).As the smaller SE6 protein also bound p300 this indicated that the binding motif is located within a region common to both proteins.

Identification of the p300-binding region within CRPVE6
As both LE6 and SE6 interact with p300, we generated subfragments of SE6 to map the interaction domain by MBP pulldown assays (Fig 2A).For better understanding, the amino acid positions are based on the respective amino acids in the full length CRPVE6 protein (LE6).The minimal interaction domain consisted of aa 166-214 (Fig. 2B).Further C-or Nterminal truncations (aa 178-214 or aa166-197) resulted in a complete loss of p300 binding.
To further narrow down the binding motif, we performed an alanine scanning mutagenesis of aa 166-178 and aa197-214 by replacing four to five consecutive residues with alanines (Fig. 2B).Quantification of the binding affinities derived from two to five individual MBP pulldown experiments revealed no effect by mutation of aa 210-214, and aa 166-169.
To verify this, we performed a co-immunoprecipitation with an HA-tagged CRPV full length E6 protein (Fig. 2C) in C33A cells, an HPV-negative keratinocyte cell line.To avoid expression of SE6 from the LE6 expression vectors the methionine at residue 98 was exchanged to serine in the wildtype (LE6M98S) and both mutant proteins (LE6M98SAla195-199, LE6M98SAla200-204).CRPVE6M98S, HA-tagged CRPVE6M98SHA, (E6M98SAla200-204), acetylated hp53 was clearly visible, indicating that the interaction of wildtype CRPVE6 with p300 is responsible for inhibition of p53 acetylation.

E6-p300 binding is important for carcinogenesis
To analyze the importance of the ability of E6 to bind p300 and inhibit the acetylation of p53 in vivo, we created p300-binding deficient (pLA2-CRPVE6Ala195-199 and pLA2-CRPVE6Ala200-204) and competent (pLA2-CRPVE6Ala210-214) E6 mutant proteins in the context of the CRPV genome.Wildtype and mutant CRPV genomes were each injected at six sites in the skin of the back of two New Zealand white rabbits.Tumor development and growth were monitored for five months.Wildtype CRPV and pLA2-CRPVE6Ala210-214 produced tumors comparable in size and growth at all injection sites in both animals (Fig. 4A; proteins was found with HPV38E6.This was notable, as HPV38 and CRPV are the only cutaneous PVs, that are able to immortalize normal keratinocytes.Consistent with the results obtained with CRPV, immortalization of primary keratinocytes by HPV38E6/E7 is dependent on the E6-p300-interaction.It was reported previously, that p53 accumulates in HPV38 E6/E7 immortalized keratinocytes and induces ΔNp73, but is unable to displace ΔNp73 from p53 responsive promoters (24).This is consistent with our findings as binding of p53 to its responsive promoters is dependent upon the acetylation state (45,48).The observed lack of immortalization of keratinocytes by a p300-binding deficient HPV38E6 could be due to acetylation of p53 by p300 which then can overcome the ΔNp73 blockage and induce senescence or apoptosis.Interestingly the cutaneous PV types behave similar to the adenoviral E1A protein, which binds to the TAZ2 domain of p300/CBP and competes with p53 for p300 binding (49).Even though the binding domain of E1A shows only little overall homology to the CRPV or HPV38 E6 p300 binding domain, all three proteins contain a hydrophobic residue followed by phenylalanine.One difference between CRPVE6 and the cutaneous HPVE6 proteins is the ability of the latter ones to bind E6AP, which makes them capable of degrading the UV-induced Bak protein to escape induction of apoptosis.For CRPV such a function is not required as the fur of the rabbit protects the skin from UVirradiation.
It has been described that genital HPV types that inactivate p53 via E6AP-dependent degradation also prevent p300-mediated acetylation of p53.However, these E6 proteins differ from CRPV and HPV38 E6 because they can bind to p53 and form trimeric complexes with p53 and p300 (28).The HPV16E6 protein interacts with the N-terminal CH1 and with Cterminal regions of p300/CBP, including the TAZ2 domain (27,29).The mechanism of p53

Figure 1 :
Figure 1: CRPVE6 represses p53 mediated p21 induction and interacts with p300 A) Cutaneous PV E6 proteins do not reduce p53 stability.Human HPV16E6/E7-, HPV 38E6/E7-or rabbit CRPV E6/E7-immortalized keratinocytes were treated with cycloheximide (CHX) or MG132 and p53 levels were analyzed at different timepoints by immunoblotting.B) RNA was isolated 48h post transfection from Saos-2 cells transiently transfected with human hp53, rabbit rp53 and CRPVE6 expression vectors.Induction of p21 mRNA was analyzed by quantitative real time PCR (** = p-value <0.005).C) E6-MBP fusion proteins purified from E. coli were incubated with HEK293T cell lysates.Eluates were analyzed by western blotting.D) upper panel: MBP-pulldown analysis of CRPVSE6 and the G38V mutant with p300.Lower panel: MBP-fusion protein input determined by Coomassie stain.Aliquots of the purified, immobilized MBP proteins were taken prior to the incubation with HEK293T lysate.

Figure 2 :
Figure 2: Identification of the p300 binding site in CRPVE6A) MBP interaction assay using SE6 fragments, wildtype SE6 proteins and MBP.B) Alanine scanning mutagenesis of CRPVSE6 identifies residues 195-204 as p300 interaction domain.The amino acids within the mutant proteins were changed to alanine at the positions (Pos.)indicated.The protein amount for the immunoblots was normalized according to protein concentration.C) Coimmunoprecipitation analysis of untagged, HA-tagged wildtype and mutant E6 with p300.

Table 1 ;
HPV16on April 14, 2017.© 2010 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on July 27, 2010; DOI: 10.1158/0008-5472.CAN-10-1307 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Copyright © 2010 American Association for Cancer Research

Table 2 )
. Multiple small tumors became visible around 25 days post infection and grew up to on April 14, 2017.© 2010 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on July 27, 2010; DOI: 10.1158/0008-5472.CAN-10-1307 on April 14, 2017.© 2010 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on July 27, 2010; DOI: 10.1158/0008-5472.CAN-10-1307