Roles for E1-independent replication and E6-mediated p53 degradation during low-risk and high-risk human papillomavirus genome maintenance
[['Papillomaviruses ( PV ) are small double-stranded DNA viruses which infect stratified epithelia , with the completion of their life cycle being intimately linked to the terminal differentiation program of keratinocytes .', 'One of the most distinctive characteristics of papillomaviruses is their genotype-specific host-restriction , and the preference shown by different papillomavirus types for distinct anatomical sites[1–3] .', 'Human papillomaviruses ( HPVs ) , which consist of more than 200 HPV types , are subdivided into 5 genera , and within the alpha genus , HPVs are further divided into low- and high-risk types based on the cancer risk associated with their infection [4 , 5] .', 'HPVs express a relatively small number of early and late viral genes during their life cycle , which are tightly regulated according to the differentiation stage of the infected keratinocytes .', 'The viral genes may be categorized as ‘core genes’ or ‘accessory genes’ .', 'All known papillomaviruses have evolved a group of core genes that were present early during papillomavirus speciation , which share similarities in sequence and protein function .', 'The core genes play primary roles in viral genome replication and amplification ( E1 and E2 ) as well as virion assembly ( L1 and L2 ) [6–10] .', 'In contrast , the accessory genes ( E6 , E7 and E5 ) , which are often referred to as viral oncogenes , have evolved in each papillomavirus type as it adapted to different epithelial niches [11–13] .', 'The sequence and function of these genes are similar yet divergent between types compared to the core genes .', 'In general , accessory genes are involved in modifying the cellular environment in order to complete the viral life cycle at the site of infection .', 'Accessory genes play a variety of important functions at different stages of the viral life cycle , such as the maintenance of infection in the basal layer and the amplification of viral genomes in the suprabasal layers .', 'As such , the function of these accessory genes as well as their regulation may largely attribute to virus diversity in tropism and pathogenicity [5 , 6] .', 'In fact , the deregulation of the accessory genes of high-risk HPV can mediate the progression to invasive cancer [4 , 14] .', 'The high-risk HPV-mediated carcinogenic process has drawn most of the research effort in the past decades , leaving open important questions about the comparative analysis of the evolutionary adaptation of different HPV types in relation to their specific epithelial sites of infection .', 'Among the best characterized functions of high-risk HPVs there are the E6-mediated degradation of p53 and PDZ domain-containing proteins and the activation of hTERT ( reviewed in [12] ) .', 'High-risk HPV E7 is known to interact and destabilise pRB family proteins , promoting cell cycle re-entry in the parabasal layers of the epithelium and above ( reviewed in [11] ) .', 'Furthermore , both E6 and E7 expressed by high-risk HPV types have multiple additional functions , such as the inhibition of differentiation , apoptosis and host-immune response .', 'In many cases , low-risk HPV E6 and E7 are considered as a ‘de-potentiated’ version of their high-risk counterparts .', 'These HPV types are maintained and propagated in the general population without having the carcinogenic properties of high-risk HPVs , indicating that the induction of cancer is a collateral effect of the life cycle strategy of high-risk HPVs rather than an adaptation .', 'In this study , we have performed a comparative analysis of how ‘core’ and ‘accessory’ proteins cooperate in promoting the replication of both low- and high-risk viral episomes .', 'We have used HPV11 and 16 as the key prototypes of low- and high-risk alpha HPV types respectively , and used the immortal and isogenic NIKS keratinocyte cell line as a cell model to draw direct comparisons of the life cycle strategies used by the two viruses .', 'We have identified a two-phase HPV replication mode common to both low- and high-risk alpha papillomaviruses that is regulated by cell density .', 'Our data shows that viral replication switches from requiring E2 and being E1-independent while genomes are maintained , to being E1-dependent as keratinocytes commit to differentiation at high cell density , a situation that also requires the function of E6 and E7 .', 'Curiously , the low-risk E6 protein was necessary in order to sustain low-risk HPV genome copy number as cell confluence was reached , and that like high-risk E6 , this was dependent on E6-mediated p53 proteasomal degradation .', 'Our results suggest that for both high and low-risk HPVs , that E1 is non-essential for synchronous HPV genome replication during maintenance , and that the low-risk E6 protein has a p53-degradation function that is regulated at cell confluence , and which like that high-risk HPV types , can inhibit commitment to differentiation and stimulate cell cycle entry .'], ['293T ( ATCC ) were maintained in Dulbecco’s Modified Eagle’s Medium ( DMEM , SIGMA ) supplemented with 10% fetal calf serum ( FCS , HyClone ) and 1% penicillin and streptomycin .', 'NIKS ( a gift from Paul Lambert , McArdle Laboratory for Cancer Research , University of Wisconsin ) , a HPV-negative spontaneously immortalised human keratinocyte cell line , was maintained at sub-confluence on γ-Irradiated J2 3T3 feeder cells ( a gift from Paul Lambert ) in F medium with all supplements as previously described [15] .', 'Bromodeoxyuridine ( BrdU ) was used at a final concentration of 30 μg/ml for 18 hours .', 'The pSPW12 plasmid was a kind gift from Prof . Margaret Stanley ( University of Cambridge ) containing the HPV16 genome .', 'The pBT-1 clone of HPV11 ( Hershey ) plasmid was a kind gift from Prof . Neil Christensen ( The Pennsylvania State University ) .', 'HPV16E6SAT , HPV16ΔPBM , HPV16 E1 defective mutant were described previously [16 , 17] .', 'All mutant genomes were constructed using a KOD -Plus- Mutagenesis Kit ( TOYOBO ) , prior to DNA sequencing to ensure that no additional base changes were present .', 'The mutants that were constructed in this study comprise the HPV16 E1 helicase defective mutant ( K383 to A ) , HPV16 E6 defective mutant ( D9 to STOP ) , HPV16 E7 defective mutant ( L13 to STOP ) , HPV16 E2 defective mutant ( G50 to stop ) , HPV11E1 defective mutant ( D46 to STOP and M47 to STOP ) , HPV11 E1 helicase defective mutant ( K384 to A ) , HPV11 E6 defective mutant ( E2 to STOP ) , HPV11 E7 defective mutant ( L15 to STOP ) and the HPV11 E2 defective mutant ( L79 to STOP ) .', 'The primer sequences used for mutagenesis are available upon request .', 'In order to construct HPV11 and HPV16 reporter genomes , two restriction enzyme recognition sites , BglII and XhoI , were generated in pSPW12 ( HPV16 ) , pBT-1 ( HPV11 ) and pSELECT–zeo-GFPBsr ( InvivoGen ) using the KOD -Plus- Mutagenesis system ( Fig 2A , primer sequences available upon request ) .', 'All mutants were sequenced to ensure that no additional base changes were present .', 'The mutated pSPW12-BglII/XhoI , pBT-1-BglII/XhoI and pSELECT–zeo-GFPBsr-BglII/XhoI were excised from the BglI and XhoI sites .', 'These two flagments , the pSPW12- BglII/XhoI plasmid or pBT-1-BglI-XhoI and GFPBsr-BglI/XhoI , were ligated using Ligation high Ver . 2 ( TOYOBO ) .', 'Plasmids containing HPV16 and HPV11 genomes ( wild type or mutant ) were digested with BamHI to release the whole viral genome .', 'The linearized HPV11 or HPV16 genomes were then re-circularized and purified as described previously , before being co-transfected with a plasmid encoding blastocidin into NIKS cells [18] .', '2x105 cells were seeded in each well of a 6-well plate the day before transfection with F-media incomplete ( no EGF ) .', 'The cells were transfected with 1600 ng of re-circularized HPV DNA , and 400 ng of pcDNA6 encoding a blasticidin resistance gene ( Invitrogen ) , using FuGENE HD ( Promega ) .', 'The next day the cells were seeded onto a 75 cm2 flask over blastcidin-resistant feeders with F-media incomplete , then NIKS cells were selected with 4 μg/ml blasticidin S with F-media complete ( with 10 ng/ml of EGF ) for 4 days and cultured extra 2–3 in the absence of Blastcidin and designated passage one ( P1 ) .', 'All experiments were carried out in triplicate using NIKS cell lines containing HPV genomes which were generated at least two independent transfections .', '3 . 3x105 NIKS cells containing HPV genomes were seeded in to a 25cm2 flask with the same number of feeder cells in F-media complete .', 'Cells were collected for analysis at day 1 , 2 , 3 , 4 and 7 .', 'All experiments were done using NIKS cells containing > than 10 copy per cell of each HPV genome at passage 2 post-transfection , .', 'The production and infection of recombinant retroviruses were accomplished as previously described [19] .', 'Construction of retrovirus vectors LXSN-HPV16E6 , HPV16E6SAT , HPV16E6ΔPDZ , HPV16E7 , HPV16E6E7 , HPV11E6 , HPV11E7 , HPVE6E7 were described previously [20] .', 'Retrovirus vectors of LXSN-HPV11E6 , HPV11E7 , and HPVE6E7 were constructed by cloning ORF of HPV11 E6 and/or E7 into LXSN using Gateway Recombination cloning technology ( Thermo Fisher Scientific ) following the manufacturer’s instruction ( primer sequences available upon request ) .', 'LXSN-11E6W133R was constructed using KOD -Plus- Mutagenesis Kit ( primer sequences available upon request ) and sequenced to ensure that no additional base changes was present .', 'The E6AP-specific shRNA constructs pCL-SI-MSCVpuro-H1R-E6APRi4 was described previously [21] .', 'To generate NIKS cells expressing E6 and/or E7 , the cells were seeded 1 day before and inoculated with at MOI of 5 in the presence of 4 μg/ml of Polybrene ( Santa Cruz ) followed by Geneticin ( Thermo Fisher Scientific ) selection ( 400 μg/ml ) for 4 days .', 'For the delivery of siRNAs , 3 . 3x105 of cells were seeded on 25cm2 flasks and transfected 12nM of siRNA using HiPerfect Transfection Reagent ( Qiagen ) at days 0 and 4 .', 'Non-targeting siRNA ( MISSION siRNA Universal Negative Control ( Sigma ) ) was used as a negative control and ON-TARGET plus Human TP53 ( Dharmacon ) was used as a siRNA to p53 .', "Total DNA from NIKS for qPCR was purified using a QIAamp DNA Mini Kit ( Qiagen ) , according to the manufacturer's instructions .", 'All samples were digested with DpnI to remove any residual input DNA prior to analysis .', "The RNA from NIKS for RT-qPCR was purified by using an RNeasy Mini Kit ( Qiagen ) , and cDNA was synthesised with SuperScript III Reverse Transcriptase ( Thermo Fisher scientific ) using 100 μM random hexamer primers , according to the manufacturer's instructions .", 'The HPV genome and GAPDH were measured by a ViiA 7 Real-Time PCR System ( Life Technologies ) using Power SYBR Green/ROX master mix ( Thermo Fisher scientific ) with 15 min denaturation at 95°C , followed by 45 cycles of 95°C for 15s and 60°C for 60s .', 'The PCR primers for qPCR were as follows .', 'The HPV11 forward primer was 5’-ACATTAGATCCGTGGACAGTACAATC-3’ or 5’-TCGTCCAGCCTAGACATTGAG-3’ , HPV11 reverse primer was 5’-TTCCTTCTTTGGTGCTTGTTGTAA-3’ or 5’- TCCAATCGTATGCATTTCCA-3’ , HPV16 forward primer was 5’-TGTTTCAGGACCCACAGGAGC-3’ , HPV16 reverse primer was 5’- CGCAGTAACTGTTGCTTGCAG-3’ , GAPDH forward primer was 5’-CCTCCCGCTTCGCTCTCT-3’ , GAPDH reverse primer was 5’-CTGGCGACGCAAAAGAAGA-3’ .', 'To quantitate HPV DNA levels in HPV genome-containing cell populations and cell lines , HPV copy number per cell was expressed relative to GAPDH copy number .', 'The human genome blast using designed primers and probe for human GAPDH indicated that there are 4 copies , comprising 2 copies of GAPDH and 2 pseudogene copies of GAPDH per NIKS cell .', 'This was confirmed by determination of GAPDH copy number against known NIKS cell number .', 'The copy number of virus genomes per cell was found to vary at passage 2 between experiments , with HPV11 WT and mutant genomes typically present at around 100 copies per cell .', 'The viral genome copy number per cell at passage 2 , day 1 was set at 100% , and subsequent changes in copy number were normalized to this .', 'The quantification of viral gene expression was carried out as described previously [18] .', '2 . 4x104 cells were seeded in each well of a 4-well culture slide ( Falcon ) and cultured for 3 and 7 days .', 'The cells were washed in PBS and fixed in 4% paraformaldehyde ( PFA ) in PBS for 30 min . at room temperature .', 'The cells were permeabilised in PBS with 0 . 1% Triton X-100 ( Promega ) for 30 min . , then washed in PBS .', 'The cells were blocked in 10% normal goat serum ( Cell Signaling Technology ) in PBS for 1 hour .', 'The antibodies used were anti-Keratin10 antibody ( dilution 1:200 , Thermo Fisher Scientific ) , anti-p53 ( DO-1 ) antibody ( dilution 1:300 , Santa Cruz ) , anti-MCM antibody ( dilution 1:100 , Abcam ) an anti-mouse Alexa 594-conjugated antibody ( dilution 1:150 , Thermo Fisher Scientific ) .', "For p53 and MCM , the signal was amplified using a Tyramide Signal Amplification Kit ( Perkin-Elmer ) , according to the manufacturer's instructions .", 'Finally , the cells were mounted in mounting medium ( Agar Scientific ) for visualization .', 'The number of cells with positive signal were quantified using ilastik software .', 'Proteins were extracted from cells using RIPA buffer and quantified using the BCA protein assay kit ( Pierce ) , before being separated on 4–12% gradient polyacrylamide-SDS-Tris-Tricine denaturing gel ( Invitrogen ) and transferred onto PVDF membranes ( Bio-Rad ) .', 'After transfer , membranes were blocked for 1 hour at room temperature in 1% milk in PBS-T ( PBS , 0 . 1% tween20 ) .', 'Blots were then incubated overnight at 4°C with the appropriate primary antibody diluted in 1% milk PBS-T .', 'Primary antibodies used were anti-HPV16E6 ( 2E-3F8 , Euromedex ) , anti-HPV16E7 ( NM2 , Santa Cruz ) , anti-p21 ( EA10 , Abcam ) , anti-p53 ( DO-1 , Santa Cruz ) , anti-GAPDH ( Millipore ) , followed by the appropriate HRP-conjugated secondary antibody ( GE Healthcare ) , and detection using ECL , or ECL plus kits ( GE Healthcare ) or by the appropriate IRDye 800CW fluorescent secondary antibody ( Licor ) followed by detection using an Odissey imaging system ( Licor ) .', 'Sub-confluent NIKS were collected and fixed for 10 min . in 4% PFA in PBS at a concentration of 1x106 cells/ml at room temperature .', 'Immediately before sorting , the cells were passed through a 40μm cell strainer ( BD Biosciences ) .', 'The cells were sorted on a Dako Cytomation MoFlo MLS high-speed cell sorter .', 'DNAs were extracted from the sorted cells as described above .', "5x104 cells were seeded in each well of a 12-well and transfected Cignal p53 Pathway Reporter Assay Kit ( Qiagen ) at the same day , according to the manufacturer's instructions .", 'The cells were cultured for 3 or 7 days and collected .', "The activities of firefly and Renilla luciferases were measured by a FLUOstar Omega Microplate Reader ( BMG LABTECH ) using Dual-Luciferase Reporter Assay System ( Promega ) , according to the manufacturer's instructions .", 'TDIG-labelled probes comprising the entire HPV11 or HPV16 genome were prepared , and Southern blot analyses were carried out using DIG High Prime DNA Labelling and Detection Starter Kit II ( Roche ) following the protocol provided by the manufacturer .', 'Briefly , digested DNA was separated on a 0 . 7% agarose gel , soaked in 0 . 25 M HCl for 15 min , and alkaline transferred onto nylon membranes ( Boehringer Mannheim ) .', 'The membranes were prehybridized in Hybrisol I ( Millipore ) for 1 h at 42°C .', 'A DIG-labelled probe was applied for hybridization , and the hybridized DNA was visualized using the detection kit .', 'DNA was mixed with cesium chloride ( CsCl ) , and the mixture was adjusted to a volume of 4 . 5 ml , and a the density of 1 . 753 g/ml ( i . e . corresponding to a refractive index of 1 . 404 ) .', 'The DNA-CsCl solution was transferred to Beckman ultracentrifuge tubes , and samples were centrifuged at 30 , 000 rpm at 22°C for more than 48 h in a SW55 rotor .', 'After centrifugation , the tube was inserted into a gradient collector , a hole was punctured at the bottom of the tube , and fractions of 5 drops each were collected in Eppendorf tubes ( up to 50 fractions ) .', 'The DNA concentration of each fraction was measured using a spectrophotometer , and the refractive index measured using a refractometer , after which the fractions were slot blotted onto a positively charged nylon membrane .', 'The wells of the slot blotter were washed with denaturation buffer ( 0 . 5 M NaOH , 0 . 5 M NaCl ) .', 'The membrane was then air dried and UV cross-linked .', 'The HPV genomes were detected using DIG-labelled probes ( see Southern blot analyses above ) .'], ['In order to compare the specific requirements for HPV16 and HPV11 genome replication in ‘infected’ basal-like keratinocytes we used NIKS cells , which are an isogenic immortal keratinocyte cell line previously shown to recapitulate the full epidermal differentiation program in vitro and to support the full HPV life cycle [22–24] .', 'In order to establish NIKS cells harboring low- and high-risk HPV genomes , HPV11 and HPV16 genomes were transfected into NIKS cells along with a plasmid encoding a blasticidin resistance gene .', 'Following drug-selection with blasticidin S , cells containing the HPV genomes were expanded and maintained at sub-confluent conditions for 2 to 8 passages .', 'Total DNA was extracted at each passage and treated with DpnI to remove the input genomes , and the levels of replicated viral genome were monitored by qPCR using GAPDH as a reference gene to calculate the HPV genome copy number per cell .', 'As previously indicated [25–27] , the HPV11 genome copy number rapidly declined upon passage to typically reach less than 0 . 2 copies/cell by passage 8 ( Fig 1A ) .', 'In contrast , HPV16 genomes , and indeed the genomes of other high-risk HPV types [23] , persisted at a uniform copy number throughout the experiment .', 'Although the actual HPV genome number varied between experiments , and was different for each HPV type , the trend was similar between passages .', 'In order to overcome these tissue culture issues , and to carry out our experiments in a more controlled manner , we decided to monitor the viral genome copy number variations within the same passage ( passage 2 ) , growing cells from sub-confluent to post-confluent conditions in a 7 day time-frame .', 'Previous studies have indicated that epidermal keratinocytes cultured in vitro switch between two interconvertible growth modes depending on the local cell density: an expanding growth mode , characterized by and excess of proliferating cells , and a balanced growth mode in which proliferation is counterbalanced by differentiation [28] .', 'Similarly , as can be seen in Fig 1B ( upper panels ) , NIKS cells grown in monolayer switch from a proliferative and undifferentiated ( expanding ) growth mode at sub-confluence ( Day 3 ) , in which they express the cell cycle progression marker MCM7 in the absence of the early keratinocyte differentiation marker keratin 10 ( K10 ) , to a differentiated and post-mitotic ( balanced ) growth mode at post-confluence ( Day 7 ) .', 'Interestingly , when low passage NIKS harboring HPV11 and HPV 16 ( passage 2 post-transfection/selection ) were analyzed in a similar way , NIKS/HPV11 showed a very similar growth pattern to that of parental NIKS Fig 1B middle panels , Fig 1C and 1D ) , indicating that low-risk HPV fails to modulate keratinocyte homeostasis .', 'In contrast however , while HPV16 did not significantly affect the growth mode of NIKS at sub-confluence , it conferred a potent proliferative capacity to post-confluent cells , which was accompanied by a loss of keratinocyte differentiation marker ( Fig 1B lower panels , Fig 1C and 1D ) .', 'This indicates that HPV16 is able to overcome the normal contact inhibition signals promoting the maintenance of an expanding growth mode at high cell densities .', 'We then went back to analysing the HPV genome copy number in low passage ( passage 2 ) HPV11/16 transfected NIKS , and found that both HPV11 and HPV16 have the same ability to maintain their genomes in sub-confluent keratinocytes ( Fig 1D; days 1–4 ) .', 'At post-confluence ( Day 7 ) , however , HPV16 genome copy number undergoes an approximately 3-fold increase relative to day 4 , while HPV11 genome copy number declined significantly ( Fig 1E ) .', 'When taken together , these data indicate that maintenance-replication of both low- and high-risk HPV types can be similarly efficient , but that HPV11 is unable to overcome keratinocyte differentiation at higher cell density , which compromises its replication ability .', 'In all cases , viral genomes were episomal , as experiments were carried out shortly after transfecting circularized virus genome ( confirmed by Southern blotting at passage 2 ( [18] , S1 Fig ) ) .', 'As predicted from this , transcript mapping studies revealed a characteristic ‘episomal’ pattern of viral gene expression in both HPV16 and HPV11-NIKS , with the majority of transcripts spanning E6 and E7 , and terminating downstream of the E5 ORF[18] .', 'As NIKS cells underwent 2 or 3 doublings during the first 4 days of our experiments , our data suggest that HPV11 does not differ from HPV16 in its overall replication competence , and can replicate along with cellular DNA in proliferating keratinocytes .', 'Importantly , our results also suggest a similar ability of both genomes to partition accurately during cell division .', 'To investigate this further , a hEF1-HTLV promoter/GFP-Blastocidin reporter cassette was inserted into the late gene region of HPV11 and HPV16 ( Fig 2A ) in order to directly visualize cells containing HPV genomes following transfection , an approach similar to the that used previously [29 , 30] .', 'The GFP-Blastocidin fusion gene also allowed the use of drug-selection to enrich for cells harbouring recombinant viral episomes .', 'This is particularly important for low-risk HPV types such as HPV11 , which does not obviously modify the cell phenotype , and has a tendency to decline in copy number over passage ( Fig 1A and 1B ) .', 'To examine whether the GFP intensity correlated with HPV copy number , NIKS cell populations harboring either the HPV11 or HPV16 reporter genomes ( i . e . NIKS/HPV11GFPbsr or NIKS/HPV16GFPbsr , respectively ) , were separated by fluorescent activated cell sorting ( FACS ) , into 4 groups according to the intensity of the GFP signal .', 'Total DNA was subsequently extracted from each group , and HPV genome copy number was quantified by qPCR .', 'In both the NIKS/HPV11GFPbsr and NIKS/HPV16GFPbsr populations , the intensity of the GFP signal correlated well with HPV genome copy number ( Fig 2B ) .', 'The NIKS/HPV11GFPbsr and NIKS/HPV16GFPbsr populations were then seeded along with parental NIKS at a ratio of 1:50 .', 'Colonies were allowed to expand in the dish for 4 days without drug selection , before being fixed and examined by fluorescent microscopy .', 'Single GFP-positive cells were readily apparent immediately after plating , with larger GFP-positive colonies developing by day 4 .', 'Although there was great diversity in the GFP signal , suggesting great diversity in viral copy number , in individual colonies , individual cells within the same colony always showed uniform levels of fluorescence , irrespective of whether they contained HPV16 or HPV11 genomes ( Fig 2C and 2D ) .', 'In fact , when normalized to the mean GFP intensity of each colony , the variation in GFP intensity amongst individual cells in both NIKS/HPV11GFPbsr and NIKS/HPV16GFPbsr colonies was broadly similar ( Fig 2E ) .', 'When taken together , these results suggest that the HPV11 genome is partitioned with similar dynamics to that of HPV16 in proliferating keratinocytes .', 'Following papillomavirus infection of basal keratinocytes , the productive phase of the virus life cycle is triggered by host keratinocyte differentiation , with viral genome replication and amplification rising gradually as cells enter the suprabasal layers , followed eventually by the expression of viral capsid proteins and virion assembly in the uppermost layers of the epithelium [31 , 32] .', 'The fact that HPV11 is unable to maintain or amplify its genome during the commitment to differentiation in our experimental setting ( Fig 1B and 1E ) , indicates that the modulation of keratinocyte differentiation is crucial for genome maintenance , and that differentiation-commitment can negatively influence the HPV replication machinery .', 'During productive infection , the viral DNA helicase E1 is essential for genome amplification in the mid epithelial layers [7] , whereas the importance of E1 for maintenance-replication in basal keratinocytes is still controversial [17 , 33] .', 'This data suggests that HPV might follow a ‘two-phase’ replication mode with respect to E1-dependency [34] .', 'To establish the contribution of E1 to HPV16/11 maintenance-replication , as well as for HPV16 genome amplification in differentiating keratinocytes , E1 deficient mutants were generated in the context of the full length HPV11 and HPV16 genomes ( HPV16E1def & HPV11E1def ) , and stable NIKS populations were established as described above .', 'At the time of seeding for the experiment , ( passage 2 post-transfection/selection ) viral genomes were predominantly episomal as determined by Southern blotting ( S1 Fig ) .', 'The viral genome copy number was monitored to see its variations within the same passage in the same way as shown in Fig 1E ) .', 'As can be seen in Fig 3A , both HPV11E1def and HPV16E1def genomes were maintained at similar levels as the WT genomes until day 4 during the ‘maintenance-replication’ phase , but declined dramatically post-confluence ( Day 7 ) .', 'These data support the hypothesis that HPV16 at least , can switch its replication mode from E1-independent to E1-dependent replication as cell density increases , and that HPV16 does not simply hamper transit to a balanced growth mode at high cell density and prolong the expanding growth mode as well as the maintenance replication of virus genome .', 'In addition , these results show that both HPV11 and HPV16 can replicate without the E1 viral helicase in expanding NIKS cells , and that the inability of low-risk HPV to overcome differentiation signals triggered by high cell densities has dramatic consequences on their replication capacities .', 'To support these conclusions , a second set of E1 defective mutants were made in the context of the HPV16 and 11 genomes , this time to disrupt the integrity of the conserved E1 helicase domain that is essential for E1’s replicative function ( HPV11 E1K484A and HPV16 E1K483A ) [35] .', 'The results obtained with these mutants were indistinguishable from those seen with HPV11E1def and HPV16E1def mutants ( Fig 3B ) .', 'To determine the two modes of HPV DNA replication in different cell density , we investigated how many times HPV DNAs replicate in a single S phase by labelling cells with BrdU [26 , 27 , 34] .', 'As Hoffman et al . reported , DNAs that have replicated once will incorporate BrdU on a single DNA strand ( heavy-light , HL ) .', 'Those that have replicated more than once will incorporate BrdU on both strands ( heavy-heavy , HH ) , and those that didn’t replicate will have no BrdU in their DNA ( light-ligh , LL ) .', 'These three DNA species were separated on a cesium chloride gradient according to their different densities , with LL DNA at 1 . 709 g/ml ( Refractive Index ( RI ) = 1 . 400 ) , and HL DNA at 1 . 753 g/ml ( RI = 1 . 404 ) and HH DNA at 1 . 795 g/ml ( RI = 1 . 708 ) ( Fig 4A and 4B ) .', 'Both HPV16WT and HPV11WT genomes were replicated once in a single S-phase , with the emergence of HL viral DNA , but not HH viral DNA at day 3 during the E1-independent maintenance replication phase ( Fig 4B ) .', 'In contrast , the HPV16 WT genome was replicated more than once at day 7 , during the E1-dependent replication phase , with the emergence of HH viral DNA at day 7 ( Fig 4B ) .', 'These results suggest that the mode of HPV genome replication switches from a synchronous mode ( i . e . once in a single S-phase ) , to amplification mode ( i . e . multiple times in a single S-phase ) in a cell-density related manner .', 'During the establishment of infection , it is thought that one single viral particle may efficiently establish the full viral intracellular life cycle [36] , suggesting that the papillomavirus DNA copy number is amplified to a certain level ( 50 to 400 ) per cell immediately after infection .', 'Prior to P1 , at 3 days post transfection , both HPV16WT and HPV11WT genomes were replicated once in a single S-phase , with the emergence of HL viral DNA but not HH , with only a small proportion of total HPV DNA contributing to replication ( Fig 4C ) .', 'At this point the majority of the viral DNA extracted from the NIKS cells is DpnI sensitive ( 91 . 2% ( 1 . 6 standard deviation ) ) .', 'Interestingly , both the HPV11 and 16 E2 defective genomes were not successfully replicated , with no HL and HH viral DNA produced ( Fig 4C ) .', 'These results suggest that in our experiments during the establishment phase , HPV genomes are replicated once per S-phase in an E2-dependent manner , after the introduction of multiple copies of viral DNA by transfection .', 'We suspect that the initial amplification phase that occurs after infection by a single virus , and which is thought to be E1-depenent , is bypassed as a result of the introduction of a high copy number of episomal viral DNA by transfection .', 'Although E1 is dispensable for the maintenance of either HPV11 or 16 genomes in dividing NIKS cells , it is clearly important for the HPV16 genome copy number elevation in post-confluent keratinocytes , with E1 disruption leading to HPV16 genome loss as cells transition to post-confluence .', 'The different abilities of HPV11 and HPV16 to replicate their genomes in keratinocytes at high cell density prompted us to investigate the interplay between the viral replicative machinery and the HPV accessory proteins E6 and E7 .', 'Recent studies indicate that HPV16 E6 has a predominant role , over E7 , in skewing basal keratinocyte cell fate towards proliferation by inhibiting differentiation in a p53- and Notch-dependent manner [20 , 37] .', 'In order to assess the potential roles of E6 and E7 functions in creating a replication-competent environment for the virus , we tested whether high-risk HPV E6/E7 proteins might complement the replicative deficiency of HPV11 at high cell densities .', 'To do this , we established NIKS cells stably expressing HPV16 E6 and/or E7 by retroviral transduction , and the expression of E6 and E7 were confirmed by western blot analysis ( S2A Fig ) .', 'The HPV11 genome was transfected into these cell lines and NIKS transduced with empty vector ( LXSN ) as a control , and , following drug selection , cells were seeded at passage 2 or 3 post-transfection for the evaluation of the viral genome copy number .', 'Strikingly , the expression of HPV16 E6 and/or E7 allowed replication of the wild-type HPV11 genome post-confluence ( Fig 5A left panel ) , which was associated with an inhibition of differentiation and p53 expression when HPV16 E6 was expressed either alone or in combination with E7 , as well as a potent increase in the cell-cycle activity post-confluence ( Fig 5B and 5C ) .', 'It is interesting to note however , that unlike E6 , HPV16 E7 exerts its function by uncoupling cell-cycle entry from differentiation as previously reported [38] .', 'In order to confirm that the expression of the viral helicase E1 was indeed necessary to mediate post-confluence HPV11 genome maintenance in the presence of HPV16 E6 and E7 , we transfected the E1-defective HPV11 genome in NIKS expressing HPV16 E6 and/or E7 and the viral genome copy number was monitored across 7 days .', 'The results of this assay are shown in Fig 5A ( right panel ) , and as can be seen , the lack of a functional E1 gene product impaired the ability of HPV16 accessory proteins to rescue HPV11 genome replication post-confluence .', 'Taken together , these data indicate that there is an interplay between the replication and accessory proteins of HPV , and that disruption of this interplay affects genome maintenance as cells transit towards a balanced growth mode and E1-dependent replication is trigger
['Introduction', 'Materials and methods', 'Results', 'Discussion']
['Human papillomaviruses ( HPV ) have genotype-specific disease associations , with high-risk alpha types causing at least 5% of all human cancers .', 'Despite these conspicuous differences , our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence .', 'During the maintenance phase , viral episomes and the host cell genome are replicated synchronously , and for both the high- and low-risk HPV types , the E1 viral helicase is non-essential .', 'During virus genome amplification , replication switches from an E1-independent to an E1-dependent mode , which can uncouple viral DNA replication from that of the host cell .', 'It appears that the viral E2 protein , but not E6 and E7 , is required for the synchronous maintenance-replication of both the high and the low-risk HPV types .', 'Interestingly , the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation , was also seen with low-risk HPV E6 , but in this case was regulated by cell density and the level of viral gene expression .', 'This allows low-risk E6 to support genome amplification , while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance .', 'Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication .', 'Despite the well-established differences in the viral pathogenesis and cancer risk , it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes , albeit with important differences in their regulatory control .', 'Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers .', 'Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance .']
['Human papillomavirus research has been prioritised towards understanding the progression of high-risk HPV infections to malignant cancer , rather than the regulation of the HPV productive cycle .', 'Low-risk HPV types are often considered as a ‘less effective’ version of their high-risk counterparts .', 'To date , they have been inadequately investigated and have generally served as reference viruses that lack oncogenic HPV functions .', 'Here we carried out a comparative analysis using the most prominent high-risk ( HPV16 ) and low-risk ( HPV11 ) HPV papillomaviruses , focusing on viral genome replication and maintenance in the epithelial basal layer .', 'During the maintenance phase , the E1 viral helicase of both HPV types are dispensable .', 'Neither E6 nor E7 proteins are required for the maintenance replication .', 'Once cells become confluent , replication switches from an E1-independent to an E1-dependent mode and requires p53 degradation by E6 .', 'Interestingly , the characteristic ability of high-risk E6 to degrade p53 and inhibit keratinocyte differentiation was also seen with low-risk HPV E6 , but in this case was regulated by cell density and the extent of viral gene expression .', 'Despite the differences in the pathogenicity of low- and high-risk HPV types , they appear to follow similar molecular strategies to complete viral life cycle .']
['medicine and health sciences', 'keratinocytes', 'pathology and laboratory medicine', 'pathogens', 'hpv-11', 'microbiology', 'cell differentiation', 'epithelial cells', 'viruses', 'developmental biology', 'dna replication', 'dna viruses', 'viral genome', 'dna', 'microbial genomics', 'viral genomics', 'papillomaviruses', 'animal cells', 'medical microbiology', 'hpv-16', 'microbial pathogens', 'biological tissue', 'viral replication', 'biochemistry', 'cell biology', 'anatomy', 'nucleic acids', 'virology', 'viral pathogens', 'genetics', 'epithelium', 'biology and life sciences', 'cellular types', 'genomics', 'organisms']