We have shown previously that Sp100 (a component of the ND10 nuclear body) represses transcription, replication and establishment of incoming human papillomavirus (HPV) DNA in the early stages of infection. In this follow up study, we show that Sp100 does not substantially regulate viral infection in the maintenance phase, however at late stages of infection Sp100 interacts with amplifying viral genomes to repress viral processes. We find that Sp100 localizes to HPV16 replication foci generated in primary keratinocytes, to HPV31 replication foci that form in differentiated cells, and to HPV16 replication foci in CIN 1 cervical biopsies. To analyze this further, Sp100 was down regulated by siRNA treatment of differentiating HPV31 containing cells and levels of viral transcription and replication were assessed. This revealed that Sp100 represses viral transcription and replication in differentiated cells. Analysis of Sp100 binding to viral chromatin showed that Sp100 bound across the viral genome, and that binding increased at late stages of infection. Therefore, Sp100 represses the HPV life cycle at both early and late stages of infection.
Human papillomaviruses (HPVs) establish a persistent infection in the cutaneous and mucosal epithelia of their hosts [1]. The virus infects the basal layer of keratinocytes through a micro-fissure and establishes a persistent reservoir of infection in these dividing cells. When the infected cells differentiate during the process of tissue renewal, late viral replication and transcription are induced, and viral particles assemble in the most superficial layers of the epithelium. This persistent, differentiation-dependent life cycle requires several different stages of viral DNA replication: immediately upon infection there is a limited amplification of viral DNA; next the viral genome must become “established” in the cell and be maintained at a low copy number as an extrachromosomal replicon for many cell divisions; and finally, the viral genome must amplify to very high levels in differentiated cells [2]. Like many other DNA viruses, the early stages of HPV transcription and replication initiate at, or adjacent to, the nuclear structure, ND10 [3]. During primary infection, the viral minor capsid protein, L2, delivers the viral DNA to the ND10 body by interaction with the PML protein, and this is important for efficient infection [4,5]. Furthermore, L2 causes reorganization of ND10 and the displacement of the ND10 factor, Sp100 [6]. In support of this finding, we have shown previously that Sp100 represses transcription of incoming HPV18 genomes [7]. During the maintenance stage of infection, levels of viral transcription and replication are not dramatically affected by the Sp100 proteins [7,8]. In cells containing extrachromosomal HPV18 genomes, we find that downregulation of Sp100 increased viral replication and transcription only ~1. 5-fold (this was not of statistical significance). Habiger et al. observed a similar increase in HPV31 transcription and replication in CIN612-9E cells, which did reach significance. Furthermore, they showed that interferon (IFN) κ induces Sp100, which in turn represses HPV31 transcription [8]. During the productive stage of the HPV lifecycle, amplification of viral DNA is coincident with epithelial differentiation [9]. This amplification event is marked by a shift in transcriptional initiation from the early to the late promoter [10]. This results in three classes of transcripts: early transcripts that utilize the early promoter and early polyadenylation site; intermediate transcripts that use the late promoter and early polyadenylation site; and late transcripts that use both the late promoter and polyadenylation site [11]. Intermediate transcripts encode E1, E2 and E4 proteins, and late transcripts encode the capsid proteins, L1 and L2. The switch between HPV early and late transcription is highly dependent on host cell differentiation, and viral DNA replication is necessary for maximal late transcription [12]. Here we examine the role of Sp100 on viral genome amplification and viral transcription during the productive stage of the viral lifecycle. We observed that Sp100 associates with replication factories formed by expression of HPV16 E1 and E2 in keratinocytes, as well as replication foci formed upon differentiation in the HPV31 containing cervical cell line CIN612-9E. Sp100 is also associated with HPV replication foci at the onset of DNA amplification in the upper layers of a cervical CIN 1 lesion. We observed that Sp100 primarily repressed late HPV31 mRNA transcription, and limited viral replication, in differentiating cells. Using chromatin immunoprecipitation, we show that Sp100 binds across the viral genome and that binding increases upon differentiation. Together, these data show that Sp100 functions as a host restriction factor at both early and late times of the HPV lifecycle.
Nuclear foci that replicate HPV DNA can be formed by coexpression of the viral E1 and E2 proteins, and these have been previously shown to localize adjacent to ND10 bodies [3]. To investigate this further and to determine if Sp100 is also recruited to sites of viral DNA replication, we generated HPV16 replication foci in a human foreskin keratinocyte (HFK) conditionally immortalized cell line, 1A, as previously described [13]. 1A cells were transfected with HPV16-E1 (pMEP9-16 EE-E1) and HPV16-E2 (pMEP4-16 FLAG-E2) expression vectors, along with either empty vector DNA (pKS), HPV16 origin-containing DNA (pKS-16Ori) or the recircularized HPV16 viral genome. The localization of the viral replication proteins with respect to PML and Sp100 was assessed by confocal microscopy. Both PML and Sp100 localized in nuclear foci (ND10 bodies), which were often observed in association with the E1/E2 foci. In the absence of a viral replicon, PML localized with E1/E2 foci in ~30–40% cells (Fig 1A, panels i-iv and Fig 1B). Introduction of replication competent DNA (pKS-16Ori or a recircularized HPV16 genome) increased the association of PML with E1/E2 foci to >95% (Fig 1A, v-viii and ix-xii; Fig 1B). This confirmed that the association of PML with the HPV replication proteins is enhanced in the presence of replicating viral DNA [3], and showed that Sp100 is also associated with HPV replication foci. PML and Sp100 associated with HPV replication foci in either a satellite association around or adjacent to E1/E2 foci, or internally within the replication factories. This internal association was seen only in the presence of replicating viral DNA and was observed more frequently in the presence of the entire HPV16 genome than the plasmid containing only an HPV16 origin (Fig 2A, 2B and 2C). To better assess whether increased internal association of Sp100 in viral replication factories inhibited viral DNA synthesis, we directly detected HPV16 DNA in the foci by FISH. Similar to the data presented in Fig 2, Sp100 was arranged in either a satellite configuration around viral DNA replication factories in cells stained for Sp100 and HPV16 DNA, or was found completely inside the entire replication factory (Fig 3A). Further, our analysis of Sp100 localization, in conjunction with 3D image processing, showed that internal Sp100 was closely associated, rather than colocalized with viral DNA in replication foci (Fig 3B and 3C). Notably, most cells that exhibited this internal Sp100 phenotype contained all available Sp100 in a single, and often largest, replication factory (Fig 3B). However, it was difficult to conclude whether Sp100 was repressing viral DNA replication in these factories due to the substantial amounts of viral DNA in these foci. To further analyze the association of PML and Sp100 with replication foci, we examined the cell line, CIN612-9E, which is derived from an HPV31-positive cervical biopsy [14] and is frequently used to study the later stages of the HPV lifecycle [9]. 9E cells were grown on glass coverslips to confluence and cultured for five days in high calcium medium to induce differentiation. This results in amplification of viral DNA in nuclear foci when detected by HPV31 FISH. In CIN612-9E cells, we observed three replication foci phenotypes: cells containing several small nuclear foci of HPV31 DNA; cells with a single large focus of viral DNA; or cells containing a mixture of the two (Fig 4A and 4B). Following differentiation, cells were fixed and combined FISH-IF was performed for HPV31 DNA and the PML and Sp100 proteins. In cells showing multiple, small, punctate FISH signals, almost 100% HPV31 DNA was either partially colocalized or adjacent to both PML and Sp100 nuclear foci (satellite association) (Fig 4C). Furthermore, PML and Sp100 staining patterns were nearly identical, indicating a high degree of colocalization between the two proteins. However, in cells with a singular large focus of viral DNA, Sp100 and PML were observed inside the replication factory in ~15% or ~35% cells, respectively, and displayed a satellite association in most of the remaining cells (Fig 4C). When Sp100 was found inside viral replication factories, PML was sometimes retained outside the foci or sometimes internally along with Sp100. In ~10% cells, large replication foci were devoid of any Sp100 staining but remained associated with PML. We speculate that Sp100 may have been degraded within these large factories to overcome the anti-viral, repressive effects of Sp100. Using CIN612-9E cells, we can study the regulation of the maintenance and the productive stages of the viral life cycle by directly measuring viral transcription and replication. To address the role of Sp100 in the maintenance phase, we assessed the effect of siRNA depletion of Sp100 on early and intermediate HPV mRNAs, and on viral genome copy number over three cell passes. 9E cells were seeded at low density and treated with either Ctrl (control) or Sp100 siRNA 24 hours later. Cells were harvested between 72 hours and 96 hours later and replated at low density. This process was repeated two more times and cellular DNA and RNA were collected at the end of pass 1 and pass 3 (Fig 5A). Levels of E6*I, E1^E4 and E2 viral transcripts and Sp100 depletion efficiency were monitored by qPCR (Fig 5B). This showed efficient downregulation of Sp100 over the three passes, but there was no consistent increase or decrease in viral mRNA after this treatment. Correspondingly, we were unable to observe reproducible changes in viral DNA copy number after three passes of Sp100 downregulation. Similarly, we concluded previously that Sp100 had only minimal effect on the levels of viral RNA and DNA in a cell line containing extrachromosomally replicating HPV18 [7]. Therefore, Sp100 does not affect viral transcription or replication in the maintenance phase of infection. Using CIN612-9E cells, we can also study the transition from the maintenance stage of the viral life cycle to the productive stage. Spink et al. demonstrated that differentiation activated the late viral promoter in CIN612-9E cells [12], and so we assessed the effect of siRNA depletion of PML and Sp100 on early, intermediate and late HPV mRNAs in the context of differentiation. Proliferating CIN612-9E cells were transfected with siRNA to Sp100, grown to confluence and cultured in medium containing 1. 5 mM CaCl2 for three days (see timeline in Fig 6A). siRNA depletion efficiency was monitored by qPCR for Sp100 using primers that detect all spliced transcripts for this gene (Fig 6B). At the end of the experiment (T = 6), Sp100 mRNA was still reduced 75–85% compared to siCtrl (Fig 6B). Differentiation was also monitored by the measurement of involucrin and filaggrin mRNA (Fig 6B). We observed a substantial inhibition of differentiation in siPML treated cells [15] and so these samples were excluded from further transcriptional analysis. Sp100 depletion did not affect involucrin levels, though filaggrin was observed to increase at later stages of differentiation (Fig 6B). Both filaggrin and involucrin are late keratinocyte differentiation markers [16], and so it is unlikely that Sp100 downregulation is globally promoting differentiation. Furthermore, it has been shown that activation of papillomavirus late gene transcription and genome amplification upon differentiation is coincident with involucrin expression [17]. However, we cannot completely rule out that Sp100 effects on differentiation are indirectly affecting viral late functions. Analysis of four different HPV31 RNA species showed small increases in early (E6*I; 1. 2–1. 5 fold), and intermediate (E2; 1. 0–1. 3 fold and E1^E4; 1. 4–2. 0 fold) in differentiated cells. However, consistently the late mRNA, L1 3590^5552, was elevated 3–12. 4 fold in cells depleted for Sp100 compared to control treated cells (Fig 6C). We conclude that Sp100 most likely primarily represses late transcription. To analyze the effect of Sp100 on HPV31 genome amplification, CIN612-9E cells were analyzed by qPCR and Southern blotting (Fig 7A and 7B). After Sp100 downregulation, the viral DNA copy number in differentiated cells was consistently increased. The magnitude of the increase was not high, probably because of the small percentage of cells that switch to the productive phase of viral replication after calcium treatment, but it was highly consistent. Taken together, these data suggest that Sp100 associates with viral replication factories to limit the viral DNA synthesized during genome amplification, and to repress transcription of late mRNAs transcribed from the newly amplified DNA. Sp100 encodes several splice variants, commonly referred to as Sp100A, Sp100B, Sp100C and Sp100HMG [18] and S1 Fig. Each of these isoforms share a common N-terminal domain that promotes dimerization [19] and another that mediates interaction with heterochromatin via association with the Heterochromatin Protein-1 (HP-1) family of proteins [20]. Additionally, the Sp100B, C and HMG isoforms share a DNA binding motif known as the SAND (Sp100, AIRE-1, NucP41/45 and DEAF-1) domain that directly binds DNA [21]. Sp100C and—HMG also have additional DNA binding (HMG box) and chromatin interacting motifs (PHD and bromodomains) that further implicate Sp100 in regulation of cellular gene expression [18]. To determine which Sp100 isoforms were responsible for repression of HPV transcription and replication, we first measured the isoforms present in primary HFKs. Using primers specific to either Sp100A, B, C or HMG, we found that all four Sp100 isoform transcripts were expressed in primary human keratinocytes (S1 Fig, panel C). Sp100A mRNA was the most abundant isoform, followed by Sp100B mRNA. Sp100C and Sp100HMG mRNA were both detectable, but at levels four to fifteen times lower than Sp100A for Sp100C and Sp100HMG, respectively. At the protein level, the Sp100 isoforms are predicted to migrate at approximately 52 kDa (Sp100A), 75 kDa (Sp100B), or 97 kDa (Sp100C and HMG). However, most studies detect only two predominant molecular weight species that migrate between 65–80 kDa and ~100 kDa when analyzed by SDS-PAGE and these species are frequently identified as either un-modified or SUMO-modified Sp100A [22]. To further characterize the Sp100 species present in HFKs, we transfected keratinocytes with expression vectors encoding EE-tagged versions of either Sp100A, B, C, HMG or an empty vector. The EE-tag adds only ten amino acids to Sp100 and thus can help delineate endogenous Sp100 isoforms. Immunoblot analysis with a pan-Sp100 antibody showed the same two predominant Sp100 species previously described (~65 kDa and ~80 kDa), as well as a low abundance of several higher molecular weight species (S1 Fig, panel D). Comparison of lysate from cells transfected with the Sp100A expression vector to the empty vector lysate revealed two major Sp100 protein species that migrated slightly above the two major endogenous Sp100 bands (S1 Fig, panel D). These are most likely unSUMOylated and SUMOylated forms of Sp100A (S1 Fig, panel D), while the species migrating slightly above 100 kDa is most likely Sp100A that has acquired two SUMO modifications [23]. Lysates from cells transfected with an Sp100B expression vector showed a major band migrating at ~100 kDa, and a minor species at ~115 kDa (S1 Fig, panel D). From the migration pattern, it seems that almost all endogenous Sp100B is SUMOylated. Lysates from cells transfected with either Sp100C or Sp100HMG expression vectors contained protein species that corresponded with the slowest migrating forms of endogenous Sp100 (S1 Fig, panel D). Because Sp100C and Sp100HMG have nearly identical molecular weights, we were unable to distinguish between them. In summary, all four major splice variants of Sp100 are present in primary human keratinocytes. Type I-IFN upregulates Sp100A and other, higher molecular weight Sp100 species in several human cell types [24–26]. Treatment of HFKs with IFN-α upregulated all Sp100 isoforms with peak induction of all isoform mRNAs six hours post-exposure (S2 Fig, panels A-F). The mRNA levels for Sp100A, -B, and -HMG increased approximately fivefold, while Sp100C mRNA levels were induced almost 10-fold over uninduced cells. We also examined the upregulation of Sp100 at the protein level following treatment with IFN. Upregulation of Sp100A and Sp100C/HMG (both un-modified and SUMO-modified) protein level was apparent 24 hours post-treatment and was sustained for at least 72 hours (S2 Fig, panel G). On examination of Sp100 in situ, we observed a substantial increase in both the size and number of PML and Sp100 nuclear foci at 48 hours post-IFN treatment (S2 Fig, panel H). These data demonstrate that the major isoforms of Sp100 are IFN-responsive in primary skin cells. Sp100A has been shown to activate a CMV-promoter controlled gene array by promoting chromatin decondensation, and adenovirus retains Sp100A in viral replication centers while excluding the SAND domain containing isoforms to evade Sp100-mediated repression [27]. Habiger and colleagues have also shown that the three longer isoforms of Sp100 can repress luciferase expression from HPV18 and HPV31 URR (upstream regulatory region) driven reporter plasmids [8]. To determine whether individual Sp100 isoforms regulate transcription and replication from the HPV genome, HFKs were coelectroporated with HPV18 recircularized genomes and each of the Sp100 expression vectors. In cells coelectroporated with an Sp100A expression vector, only a small (and non-significant) reduction in HPV18 E1^E4 or E6*I transcription was observed (Fig 8). However, the SAND domain containing isoforms (B, C and HMG) reduced viral transcription by approximately 50–60%. A mutation in the DNA binding region of the SAND domain of Sp100B reduced transcriptional activity similar to empty vector (Fig 8A); however, as shown in S1 Fig, panel D, the steady state levels of the mutated Sp100B was less than that of wild type, making it difficult to conclude that the SAND domain is crucial for the observed repression. We also analyzed viral replication in the same transfected samples. No measurable change in HPV18 replication was apparent in cells cotransfected with Sp100A (Fig 8B), but the SAND domain containing isoforms of Sp100 repressed viral replication. Furthermore, in cells transfected with the expression vector encoding Sp100B containing the SAND domain DNA binding mutation, replication was restored to wild-type levels (Fig 8B). However, as described above, we could not absolutely conclude that repression required the SAND domain function because of reduced stability of the protein. In conclusion, the three longer isoforms of Sp100 repress HPV transcription and replication. Since the repressive forms of Sp100 contain both DNA binding and chromatin binding domains, we sought to determine whether Sp100 binds to viral chromatin to repress transcription and replication in differentiated 9E cells. Primers were designed to amplify different regions across the HPV31 genome, and those that passed qPCR quality control are shown in Fig 9A. To ensure that immunoprecipitation by the Sp100 antibody was specific, we treated confluent 9E cells with interferon to increase Sp100 expression (S3 and S4 Figs). This showed that the Sp100 isoforms are upregulated by interferon in confluent CIN612-9E cells (S3 Fig, panel A), in a pattern very similar to that of primary HFKs (S2 Fig). Chromatin was prepared from growing, confluent, and calcium treated 9E cells, as well as confluent 9E cells that had been treated for 24 hours with 25 ng/ml IFNα. Interferon increased the amount of Sp100 binding to HPV31 DNA by about threefold, indicating that the ChIP assay was specific. Additional controls using negative control antibodies (IgG), and positive control histone H3 antibodies are shown in S4 Fig. This showed that the Sp100 antibody coimmunoprecipitates up to 70-fold more viral DNA than the IgG negative control. To further prove the specificity of the Sp100 antibody, Sp100 protein was downregulated by siRNA before ChIP analysis. This showed that there was a very substantial decrease in the amount of viral DNA immunoprecipitated after siSp100 treatment (S4 Fig panels B and C). Therefore, the Sp100 ChIP assay is robust, and specific. Collectively, the ChIP analyses showed that Sp100 bound to all regions of the viral genome (similar to histone H3); no specific region of the genome was targeted by Sp100 and so its repressive effect is viral sequence independent. We compared Sp100 binding to the HPV31 genome in growing, confluent, or differentiated 9E cells (treated for three days in high CaCl2 medium after reaching confluence). Viral DNA copy number increased when cells reached confluency but did not increase further after culture in CaCl2 medium (Fig 9). We saw a pronounced increase in the amount of viral DNA coimmunoprecipitated with Sp100 as cells progressed from growing to confluence, and again after CaCl2 treatment. When the percentage of genome bound by Sp100 was calculated (% input), it was observed that Sp100 binding increased both when viral DNA was amplified at cellular confluence and when differentiation was further increased after CaCl2 treatment. Overall, there was a 2. 6 to 4. 9 fold increase in Sp100 binding per viral genome upon differentiation (Fig 9). Therefore, Sp100 binding to the HPV genome increases as cells transition into the late, productive stage of infection. To obtain a more exact quantitation of the increase in Sp100-viral DNA association upon differentiation, we performed an absolute quantification of Sp100 protein and HPV31 DNA in chromatin immunoprecipitates from growing and differentiated 9E cells. The Sp100 protein content in input and immunoprecipitated chromatin samples was calculated by reference to a standard curve of in vitro translated Sp100 protein, and the absolute quantity of HPV31 genomes in each sample was measured through ChIP-qPCR analysis of input DNA and immunoprecipitated viral DNA against an HPV31 plasmid DNA standard curve. As shown in S5 Fig, there was ~ eightfold increase in the number of HPV31 genomes associated with Sp100 in differentiated cells, taking into account decreased Sp100 protein and increased viral DNA levels observed in input chromatin fractions after differentiation. Sp100 binding to viral DNA also increased substantially with interferon treatment (S4 Fig). The viral DNA copy number showed only a small decrease after IFN treatment, as measured from input chromatin samples (S4 Fig) and so the observed increase in binding reflected increased Sp100 binding per viral genome. The increase in binding in response to IFN treatment supports the hypothesis that Sp100 binding to HPV31 DNA is an anti-viral response. Interestingly, the amount of histone H3 bound to viral DNA increased with differentiation. This could be due to increased chromatin accessibility in the rapidly amplifying viral DNA in replication foci, or changes in topological restraints, as the genomes switch from bidirectional theta replication to recombination-directed replication [28]. We have previously shown that there are different patterns and enrichments of modified histones in the replication foci in 9E cells [13] and follow-up studies will analyze the role of chromatin dynamics in late viral replication. For Sp100 to repress late HPV transcription, it must be expressed in cells that amplify viral DNA and transcribe late mRNAs. Nakahara and Lambert examined PML bodies in organotypic raft tissue and showed that the number of bodies were increased in HPV-infected cells and tissue [29]. They showed that the PML bodies were present in basal and suprabasal cells, but were no longer present in the most differentiated cells. To examine if the same is true for Sp100, we stained sections of tissue derived from an HPV16-infected cervical lesion with antibodies to Sp100 and E4, and combined this with FISH for viral DNA. We noted that, as described for PML by Nakahani and Lambert, Sp100 expression was increased in HPV-infected tissue compared to normal tissue. Foci of Sp100 were observed in basal cells and these increased throughout the stratum spinosum, but disappeared in the most differentiated cells. S6 Fig shows single cell quantitation of Sp100 expression in normal and HPV16-infected cervical tissue that confirms these findings. Viral DNA amplification occurs in the upper layers of HPV16-infected tissue and we wanted to determine whether this occurs in cells that still express Sp100. As shown in Fig 10A, viral DNA amplification was initiated in cells that still contained Sp100 foci. In the layer of cells above this, full-blown DNA amplification could be observed and Sp100 was no longer present. A careful analysis and 3D reconstruction of cells initiating viral DNA amplification in the presence of Sp100 showed that in the majority of cells (~90%) Sp100 foci were localized adjacent to, or engulfed by, HPV replication foci (Fig 10B and 10C). Therefore, Sp100 foci and viral DNA replication foci interact at late times of infection, concomitant with viral DNA amplification and late gene transcription.
In this study, we found that HPV replication factories generated by expression of the E1 and E2 replication proteins, and those formed in differentiated cells containing the HPV genome, are associated with PML and Sp100 containing ND10 bodies. The ND10 bodies were mostly observed adjacent to, or in a satellite pattern around, the replication factories; however, PML and Sp100 proteins could also be observed inside large foci in close association with amplifying viral DNA. In cells containing E1-E2 generated replication foci, the normal, nuclear body staining pattern of Sp100 and PML was disrupted, and in many cases all nuclear Sp100 had accumulated into a single replication factory. Sp100-containing nuclear foci were also detected adjacent to viral replication foci in the upper layers of HPV-infected cervical lesions, and often single or multiple Sp100 foci were engulfed in a cloud of viral DNA. The role of Sp100 was analyzed in detail; we determined that Sp100 was associated with viral chromatin, and this association increased concomitant with the formation of replication foci in differentiated cells, resulting in repression of viral transcription and replication. Transient expression of the individual isoforms of Sp100 showed that the three longer isoforms of Sp100 (Sp100B, C and HMG) could limit viral replication and transcription. Sp100 proteins limit the transcription and replication of many viruses, often by repressing incoming genomes at the early stages of infection [7,8, 26,27,30–35]. The replication factories that form in the late, productive phase of several viruses are also associated with ND10 bodies and late viral functions can also be specifically repressed by Sp100 [30,36,37]. We have shown previously that Sp100 represses viral transcription, replication and establishment at early times after infection [7], and here we extend these findings to show that the differentiation-dependent, productive stage of HPV infection is also limited by Sp100. Analysis of ND10 protein modulation of viral processes can be challenging because different components can have opposing effects [37,38], and many viruses encode antagonists that negate anti-viral effects [38]. Moreover, both PML and Sp100 encode several different isoforms that differentially modulate viral transcription and replication. An additional challenge in studying the late phase of the HPV life cycle is that it is dependent on differentiation of the host keratinocyte. We successfully downregulated Sp100 in differentiated CIN612-9E cells, but siRNA-mediated repression of PML expression inhibited keratinocyte differentiation and so we were unable to further analyze the role of PML in differentiated cells. We also observed some effect of siRNA-mediated repression of Sp100 on filaggrin, but not involucrin, keratinocyte differentiation markers, and so there remains the possibility that some of the effects of siSp100 observed on late viral functions are indirect. Calcium induction of differentiation in HPV31 containing CIN612-9E cells induces viral DNA amplification and late gene transcription, though it is not possible to achieve the magnitude of viral DNA and RNA observed in vivo. Nevertheless, enough viral DNA amplification and late gene expression are induced to allow the effect of Sp100 to be measured. Repeated experiments showed that Sp100 depletion had only minimal effect on viral early or intermediate mRNA transcripts, but consistently upregulated late L1 transcripts and increased viral DNA amplification (Figs 6 and 7). This identifies a role for Sp100 in the productive stages in the viral lifecycle. Similarly, Sp100 has an anti-viral effect at both early and late stages of infection by hCMV [30], and HSV [39]. Reactivation of EBV lytic infection also results in the association of EBV genomes and viral gene products with ND10 body components [36]. In the current study, we also observed Sp100 binding to viral chromatin and showed that this binding increased with differentiation. Thus, Sp100 likely functions as a repressor of late viral transcription and replication, providing infected cells with a late-stage anti-viral activity. We have previously shown that depletion of Sp100 during maintenance replication has only minimal effects on viral DNA copy number or viral transcription [7], and we confirm that finding here for HPV31 containing-cells. Thus, Sp100 repression seems to be mostly confined to early and late stages of viral genome amplification. We speculate that this could be due to the unlicensed, unscheduled viral DNA replication that takes place at these stages. Viral DNA amplification is closely associated with the cellular DNA damage and repair response (DDR) [40], and components of the DDR interact with or localize to ND10 [41–43]. The host DDR is important for amplification of viral DNA in differentiated cells [44,45] and the recruitment of DNA repair factors to replication factories within these cells is thought to facilitate viral DNA replication in cells that have exited the cell cycle and are no longer replicating the host DNA [28,46]. This suggests a complex relationship between late-stage HPV genome amplification, the DDR, and the intrinsic immune system. We also speculate that only viral transcription from templates that have undergone amplificational replication are subject to significant Sp100 repression. This would explain why early viral mRNA transcripts (such as E6*I) are repressed at early stages of infection [7], while primarily late transcripts (L1) are repressed in differentiated cells (this study). We further examined the HPV-Sp100 interaction by characterizing the Sp100 isoforms expressed in primary keratinocytes to elucidate which isoforms could contribute to Sp100-mediated repression of HPV infection. We observed all four major Sp100 isoforms expressed in primary HFKs with Sp100A being the most abundant isoform at both the RNA and protein level (S1 Fig). Furthermore, IFN-α enhanced expression of all four isoforms (S2 Fig) in a pattern very similar to that described for BJ fibroblasts [26]. In both cell types, Sp100C was expressed at relatively low levels compared to the other isoforms but was most strongly induced at the transcriptional level by IFN-α. Habiger et al. , also recently showed that most Sp100 isoforms are expressed at reduced levels in CIN 612-9E cells, compared to primary HFKs [8], consistent with the global downregulation of interferon-induced genes in HPV-infected cells [47]. Notably, all Sp100 isoforms observed by immunoblot analysis could be induced by exogenous IFN in HPV31-containing differentiated 9E cells (S3 Fig). We also observed an increase in the SUMOylation of Sp100A upon differentiation (S3 Fig). However, we were unable to analyze the role of the Sp100 isoforms in differentiating CIN612-9E cells (siRNAs against the individual isoforms showed substantial crosstalk). Nevertheless, by cotransfecting expression vectors for each isoform along with an HPV18 viral genome into keratinocytes, we found that expression of the SAND domain containing isoforms Sp100B, C and HMG significantly reduced viral gene expression and DNA replication (Fig 8). Mutation of the DNA binding domain of Sp100B (Sp100B-Q) abrogated this repression, but this result has to be interpreted with caution as the mutated protein had decreased steady state levels. A similar result was obtained by Habiger and colleagues who found that the three longer forms of Sp100 could repress transcription from an HPV URR driven reporter plasmid [8]. Correspondingly, the SAND domain containing isoforms of Sp100 also function as repressors of HSV immediate-early transcription [25,26] and adenovirus infection [27]. In the latter case, Sp100A is recruited to adenoviral replication centers, while the repressive isoforms (B, C and HMG) are redistributed away from these sites [27]. Sp100A can decondense viral chromatin and activate viral transcription in the context of CMV infection [48], while Sp100B binds DNA and represses transcriptional activity of DNA containing unmethylated CpG motifs [49,50]. Therefore, the different Sp100 isoforms differentially regulate transcription and replication of several viruses. All four isoforms of Sp100 contain a motif that promotes interaction with ND10 bodies, but the three longer isoforms contain additional domains that bind DNA and chromatin [49,51]. This would be consistent with our finding that Sp100 colocalizes with, and binds to, viral chromatin. We did not observe specific association with any region of the viral genome and this could indicate that Sp100 is marking the genome for silencing rather than functioning at specific transcriptional elements or the viral replication origin. It has previously been shown that PML containing ND10 bodies are increased in the poorly differentiated layers of HPV-infected organotypic rafts compared to uninfected rafts, but are absent in the most superficial, differentiated layers [29]. Sp100 containing ND10 bodies have also been detected in HPV33-infected cervical tissues and similarly are absent in the upper layers of tissue [6]. This raised the question as to whether Sp100 was actually present in differentiating cells that are amplifying viral DNA. However, we show that there is a clear transition zone where Sp100 is still expressed and viral DNA amplification begins. Furthermore, nuclear foci of Sp100 (likely ND10 bodies) are very frequently located adjacent to, or engulfed by, the replicating DNA. In summary, Sp100 associates with replication foci at late times in infection and appears to repress this stage of infection by binding to viral chromatin. We have concluded that Sp100 functions as a repressor of late viral transcription and replication to provide infected cells with a late-stage anti-viral activity. However, there is also the strong possibility that HPV usurps the anti-viral functions of Sp100 to modulate the viral lifecycle. For example, Sp100-mediated repression could promote the shift from early infection to the maintenance phase of infection. Repression and condensation of viral chromatin at the late stage of infection could also prepare viral genomes for packaging in virion particles. Most likely, Sp100 attempts to restrict late viral infection, but HPV antagonizes this process, and probably hijacks some of its functions. This could explain the relatively modest effects of Sp100 we observe on late viral replication and transcription. In summary, we have shown that Sp100 represses viral replication and transcription at early and late stages of infection. Therefore, the host intrinsic immune system functions to restrict viral activity in newly infected cells, but is also activated when persistently infected cells switch into the productive stage of the viral life cycle upon differentiation. We predict that this is linked to the unscheduled viral DNA amplification that takes place at these times.
Wildtype genomes for HPV16, HPV18 and HPV 31 cloned in prokaryotic vectors have been described previously [52–54]. The mammalian expression vectors for N-terminally glu-glu (EE) epitope tagged HPV16 E1 (pMEP9-HPV16EE-E1), and N-terminally FLAG epitope tagged HPV16 E2 (pMEP4-HPV16FLAG-E2) have been described previously [55,56]. pKS (-) and pKS-HPV16 ori plasmids have been described previously [57]. EYFP-tagged versions of Sp100A, Sp100B, Sp100C, Sp100HMG, and Sp100B-W655Q were obtained from Susan Janicki (Wistar Institute; Philadelphia, PA) and have been described previously [48]. N-terminal, EE epitope-tagged versions of each Sp100 isoforms were generated by restriction digestion of pEYFP-Sp100A, pEYFP-Sp100B, pEYFP-Sp100C, pEYFP-Sp100HMG, pEYFP-Sp100B-W655Q with NheI and XhoI to remove the N-terminal EYFP tag and it was replaced with a synthesized DNA sequence containing a T7/SP6 promoter, β-actin mRNA leader sequence, and an EE epitope tag (GeneART; Hamburg, Germany). The resulting plasmids were named pCMV-TnT-Sp100A, -Sp100B, -Sp100C, -Sp100HMG and -Sp100B-W655Q, respectively. To obtain recircularized HPV genomes, 10 μg HPV DNA, cloned in a prokaryotic vector, was digested with a restriction enzyme to release the viral DNA. Following digestion, the restriction enzyme was either heat inactivated or purified away from the digested DNA using HiPure PCR Purification Columns (Roche AG; Mannheim, Germany). The released viral DNA was resuspended at 10 μg/ml in 1X ligation buffer (66 mM Tris-HCl, pH 7. 5,5 mM MgCl2,5 mM DTT, 1 mM ATP) with 40 units of T4 DNA ligase (New England BioLabs; Ipswich, MA) in a total volume of 900 μl and incubated at 16°C for 12–18 hours. Following ligation, NaCl was added to the solution to a final concentration of 0. 5 M and the DNA was precipitated with 0. 6 volumes isopropanol. Precipitated DNA was washed two times with 70% EtOH and resuspended in 1X TE buffer. The efficiency of viral genome religation was determined by separation of 200 ng pre- and post-ligation using DNA agarose gel electrophoresis. The final dsDNA concentration was determined by spectrophotometric analysis using the Nanodrop-1000 (Life Technologies; Forest City, CA). The conditionally immortalized human foreskin keratinocytes (HFK1a) was developed in our laboratory and has been described previously [56] and was cultured in F-medium supplemented with the Rho-kinase inhibitor, Y-27632 (Tocris Bioscience; Bristol, United Kingdom) at a final concentration of 10 μM. CIN612-9E cells, a cell line derived from a CIN1, HPV31-positive patient biopsy, that harbors extrachromosomal HPV31 genomes [9], were grown in F-medium without antibiotics and was provided by Lou Laimins (Northwestern University). Primary human keratinocytes were isolated from neonatal foreskins, as described previously [58]. Keratinocytes from each donor were independently designated HFK strain #. Cells were cultured in Rheinwald-Green F-medium (3: 1 Ham’s F12/DMEM-high glucose, 5% fetal bovine serum, 0. 4 μg/ml hydrocortisone, 8. 4 ng/ml cholera toxin, 10 ng/ml epidermal growth factor (EGF), 24 μg/ml adenine, and 6 μg/ml insulin) on a layer of lethally irradiated NIH J2 3T3 murine fibroblasts [59]. Antibiotics were not used unless otherwise noted. NIH J2 3T3 mouse fibroblast cultures (obtained from Craig Meyers, Penn State University) were maintained at low passage and cultured in DMEM/10% newborn calf serum. Prior to coculture with human keratinocytes or CIN612-9E cells, feeders were exposed to 60 grays of γ-irradiation using a Model 30 J. L Shepard Cs-137 Mark I irradiator (San Fernando, CA). For differentiation assays, CIN612-9E cells were transferred to low calcium basal medium (Lonza Corporation; Walkersville, MD) supplemented with SingleQuots for keratinocytes (Lonza Corporation; Walkersville, MD) containing bovine pituitary extract, hydrocortisone, and epidermal growth factor, grown until confluent, and then cultured for 96 hours in basal medium Lonza Corporation; Walkersville, MD), supplemented with 1. 5 mM calcium chloride (Lonza Corporation; Walkersville, MD). To induce Sp100 expression in ChIP assays, cells were cultured for 24 hours with 25 ng/ml carrier-free, human interferon α A (PBL Assay Science; Piscataway, NJ) in low calcium basal medium described above. HFK 1A cells cultured on glass coverslips in F-medium supplemented with 10 μM Y-27632 were transfected with HPV16 E1 and E2 expression vectors (400 ng each) and 50 ng of pKS (empty vector), pKS-16Ori, or recircularized HPV16 genome. DNA was introduced into cells using Fugene 6 (Promega Corporation; Madison, WI). Fugene 6 was mixed 3: 1 (volume/mass) with target DNA in serum-free Ham’s F-12 nutrient mixture for approximately 30 minutes at room temperature. Following incubation, lipid: DNA complexes were added dropwise to cells and incubated until cell harvest. E1 and E2 expression was induced 24 hours after DNA transfection with 3 μM CdSO4 for four hours and cells were fixed with 4% PFA for confocal microscopy analysis by indirect immunofluorescence. For cotransfection of HPV18 DNA with Sp100 isoform expression vectors, 2 μg HPV18 DNA and 1 μg Sp100 isoform DNA was incubated with 100 μl of room temperature Amaxa Nucleofection solution for 5 minutes as described in Fig legends. 106 cells were gently mixed with the DNA/Nucleofection solution and transferred to an electroporation cuvette. Electroshock was delivered using the pre-programed setting, T-007. Cells were transferred to recovery medium (F-Medium with 10% fetal bovine serum and no EGF) and plated onto irradiated murine J2/3T3 fibroblasts. For RNA extractions, CIN612-9E cells were plated at a density of 1. 3-8x104 cells/cm2 onto plates containing 1. 3–1. 5 x104 cells/cm2 of lethally irradiated J2/3T3 fibroblasts and cultured overnight. For ChIP experiments, CIN612-9E cells were cultured at a density of 4000 cells/cm2 on 15 cm plates containing 1. 3–1. 5 x104 cells/cm2 of lethally irradiated J2/3T3 fibroblasts. Pools of four siRNA duplexes that target PML (L-006547-00-0010), Sp100 (L-015307-00-0005) or a non-targeting control siRNA (D-00181a0-10-20) were purchased from Dharmacon (GE Healthcare). siRNA transfections were performed as per manufacturer’s protocol. Briefly, siRNAs were complexed with Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) in OptiMEM and added to cells with fresh F-medium at a final concentration of 20 nM. Cells were incubated with siRNA for indicated time periods before harvesting or further experimentation. All siRNA sequences are shown in S2 Table. For RNA extraction from keratinocyte coculture experiments, feeder cells were removed using Versene (Life Technologies; Forest City, CA) prior to harvest. Total RNA was isolated using the RNeasy Mini-RNA extraction kit (Qiagen; Germantown, MD). RNA concentrations were determined using the NanoDrop 1000 spectrophotometer (Life Technologies; Forest City, CA). RNA integrity analysis was performed by capillary electrophoresis using RNA 6000 Nano kits (Agilent Technologies; Forest City, CA) on a 2100 Bioanalyzer system (Agilent Technologies; Forest City, CA). Reverse transcription reactions were performed with the Transcriptor First-Strand Synthesis kit (Roche AG; Mannheim, Germany) using 1 μg of total RNA, 60 μM random hexamers, and 2. 5 μM oligo-dT primers and expression of the indicated genes was analyzed by qPCR using an ABI Prism 7900HT Sequence Detector or the QuantStudio 7 Flex Real Time PCR System (Applied Biosystems; Forest City, CA) using SYBR green PCR master mix (Roche AG; Mannheim, Germany). Each reaction mixture contained 1× SYBR green master mix, cDNA from 1 μg of RNA, and 0. 3 μM each oligonucleotide primer in a total volume of 20 μl. In each run, a 10-fold dilution series (2. 5x105-2. 5x10-2 fg) of the target mRNA standard was included to generate a curve of threshold cycle (Ct) versus log10 quantity (fg). DNA standards for the HPV31 mRNAs E1^E4 (nt 857–877^3294–3296), E6*I (nt 186–210^413–416) and L1 (nt 3562–3590^5552–5554) were commercially synthesized and cloned into the SfiI site of pMAT (GeneART; Hamburg, Germany). A DNA standard for the intermediate HPV31 E2 mRNA (nt 862–877^2646–2648) was generated by PCR amplification of HPV31 nucleotides 862–877^2646–2712 from cDNA derived from an HPV31-containing cell line and ligation of the PCR product into the cloning vector, pCR2. 1, using a TopoTA cloning kit (Life Technologies; Forest City, CA). DNA standards containing amplicons for the cellular mRNAs Sp100A, Sp100B, Sp100C and Sp100 HMG were commercially synthesized and cloned into the SfiI site of pMAT (GeneART; Hamburg, Germany). DNA standards for the cellular mRNAs of involucrin, filaggrin and cyclophillin A were generated by PCR amplification of involucrin, filaggrin and cyclophilin A cDNAs derived from primary human keratinocytes using commercially available primers from Qiagen (Germantown; MD). pRSV-neo was purchased from ATCC (Manassas, VA) and has been described previously [60]. pDRIVE-GAPDH and pCMV-SPORT6-TBP were purchased from OpenBiosystems (Pittsburgh; PA). The β-actin-GFP plasmid containing the human β-actin promoter upstream of the actin-GFP sequence was purchased from AddGene (Cambridge; MA) and has been described previously [61]. Gene expression was measured and the data are reported as the quantity of specific cDNA present in cDNA prepared from 12. 5–50 ng of RNA (labeled as quantity of RNA). The specificity of each primer pair was determined by dissociation curve analysis and the presence of single amplicons was confirmed by capillary electrophoresis using DNA1000 LabChips (Agilent Technologies; Forest City, CA) on a 2100 Bioanalyzer system (Agilent Technologies; Forest City, CA) or standard gel electrophoresis using 2. 5% agarose-Tris acetate EDTA (TAE). Primer sequences are shown in S1 Table. For all keratinocyte coculture experiments, feeder cells were removed using Versene (Life Technologies; Forest City, CA) prior to harvest. Total cellular DNA was isolated from keratinocytes using the DNeasy Blood and Tissue Kit (Qiagen; Germantown, MD) at specific times of infection. 1 to 5 ng of DNA was analyzed by qPCR using 300 nM primers and SYBRgreen Master mix (Roche AG; Mannheim, Germany). Reactions conditions consisted of a 15 minute 95°C activation cycle, 40 cycles of 10 s 95°C denaturation and 30 s 60°C annealing and elongation. Copy number analysis was completed by comparing the unknown samples to standard curves of linearized HPV18 DNA. The DNA copy number of β-actin was used as an endogenous control for all DNA qPCR experiments. qPCR for HPV31 DNA copy number was performed following isolation of DNA from CIN612-9E cells. DNA was digested with HindIII and BamHI and amplified using HPV31-specific primers under the reaction conditions described above. All qPCR was performed using an ABI 7900HT PCR system or the QuantStudio 7 Flex Real Time PCR System (Applied Biosystems; Forest City, CA). Primers for all PCR reactions are listed in S1 Table. For all keratinocyte coculture experiments, feeder cells were removed using Versene (Life Technologies; Forest City, CA) prior to harvest. Total DNA was harvested using the DNeasy Blood and Tissue Kit (Qiagen; Germantown, MD). At least 1 μg total DNA was digested with either a single-cut linearizing enzyme (HPV16, BamHI; HPV31, HindIII) for the HPV genome, or with a non-cutter (HPV16, HindIII; HPV31, BamHI) to linearize cellular DNA. After digestion, samples were separated on 0. 8% agarose-TAE gels. DNA was visualized with 0. 5 μg/ml ethidium bromide and was transferred onto nylon membranes using a Turbo Blotter downward transfer system (GE Healthcare; Pittsburg, PA). Membranes were UV cross-linked (120 mJ/cm2), dried and blocked with hybridization buffer (3X SSC, 2% SDS, 5X Denhardt’s solution, 0. 2 mg/ml sonicated salmon sperm DNA) for 1 hour. After blocking, the membrane was incubated overnight with 25ng (32P) -dCTP labeled probe (specific for HPV16 or HPV31) in hybridization buffer (3X SSC, 2% SDS, 5X Denhardt’s solution, 0. 2 mg/ml sonicated salmon sperm DNA). Hybridized DNA was visualized and quantitated by phosphor-imaging on a Typhoon Scanner (GE Healthcare; Pittsburgh, PA). To generate HPV-specific radiolabeled probes, linear viral DNA was cleaved from vector sequences and gel-purified using QiaQuick Gel Extraction kits (Qiagen; Germantown, MD). Radiolabeled probe was generated from 50 ng gel-purified linear HPV16 or HPV31 DNA with the Random Prime DNA Labeling Kit (Roche AG; Mannheim, Germany). Cells were cultured on #1. 5 18 mm glass coverslips and fixed with 4% paraformaldehyde/ PBS. Fixed cells were permeabilized with 0. 1% Triton X-100 in PBS and blocked in 5% (v/v) normal donkey serum (Jackson Immunoresearch; West Grove, PA). All primary antibody incubations were performed at 37°C for 1 hour. Antibodies used were: goat anti-PML A-20 (Santa Cruz Biotechnology, Dallas, TX; dilution 1: 50); rabbit anti-PML (Sigma Corporation; St. Louis, MO; dilution 1: 50); mouse anti-PML, PG-M3 Santa Cruz Biotechnology, Dallas, TX; dilution 1: 50); rabbit anti-Sp100 (Sigma Corporation; St. Louis, MO; dilution 1: 500); chicken anti-EE (Bethyl Laboratories; Montgomery, TX; dilution 1: 100); mouse anti-FLAG M2 (Sigma Corporation; St. Louis, MO; dilution 1: 500). Following primary antibody incubation, coverslips were washed three times in PBS. All secondary antibody incubations were performed at 37°C for 30 minutes. Alexa 488, Alexa 564, Alexa 594, Rhodamine Red-X or Alexa 647 conjugated to the desired species’ immunoglobulin was purchased from Jackson Immunoresearch (West Grove, PA) and were used at 1: 100 dilution. Coverslips were mounted using 10 μl ProLong Gold with DAPI (Life Technologies; Forest City, CA). Cells were visualized using a Leica TCS-SP5 laser scanning confocal microscope (Leica Microsystems; Buffalo Grove, IL). Cells were fixed and stained as described for IF. After antigen staining, cells were fixed again with methanol: acetic acid (3: 1, v/v) at room temperature for 10 minutes followed by 2% PFA for 1 minute at room temperature to fix the antibodies in place. Cells were treated with RNace-iT cocktail (Agilent Technologies; Forest City, A). at a dilution of 1: 1000 for 1 hr at 37°C and subsequently dehydrated in 70%, 90%, and 100% ethanol for 3 minutes each and air dried. HPV16 or 31 DNA FISH probe was prepared using the FISH-Tag DNA Multicolor Kit labeling kit (Life Technologies; Forest City, CA). DNA was conjugated to either Alexa 488 or Alexa 555, according the manufacturer’s protocol. Hybridization was performed overnight in 1X Hybridization Buffer (Empire Genomics; Buffalo, NY) with 20–75 ng of labeled probe DNA at 37°C. Slides were washed at room temperature with 1X phosphate-buffered detergent (PBD) (MP Biosciences, Inc; Cleveland, OH), followed by 1X wash buffer (0. 5X SSC, 0. 1% SDS) at 65°C, and a final wash with 1X PBD at room temperature. Coverslips were mounted using Prolong Gold with DAPI (Life Technologies; Forest City, A). and analyzed by confocal microscopy. For all keratinocyte coculture experiments, feeder cells were removed using Versene (Life Technologies; Forest City, A) prior to harvest. Whole cell lysates were extracted from primary human keratinocytes using SDS-extraction buffer (SDS-EB) (50 mM Tris, pH 6. 8; 10% glycerol, 2% SDS) supplemented with cOmplete ULTRA protease inhibitor cocktail (Roche AG; Mannheim, Germany). 1X SDS-EB was added to each sample (200 μl/3. 8 cm2 of culture dish area) and the lysate was collected by scraping. To shear genomic DNA, samples were sonicated using a cup horn sonicator (Sonics & Materials; Newtown, CT) at 15% amplitude for 1–2 minutes. Following sonication, samples were heated to 95°C for 5 minutes, and then immediately cooled to room temperature. Protein concentration of each sample was determined using the Pierce BCA Assay Reagent (Thermo Fisher Scientific; Waltham, MA). Protein samples were normalized to the same concentration using 1X SDS-EB, and 10–25 μg total protein was supplemented with 50 mM DTT and 4X LDS sample buffer to a volume of 25 μl. Samples were heated to 70°C for 10 minutes, cooled to room temperature, and separated by SDS-PAGE on 4–12% NuPage gradient gels (Life Technologies) in 1X MOPS buffer for 1–2 hours at 185V. Proteins were transferred overnight onto PVDF membrane (EMD Millipore; Stafford, VA) and subsequently immunoblotted. Primary antibodies used were: rabbit anti-PML (Bethyl Laboratories; Montgomery, TX; dilution 1: 1000); rabbit anti-Sp100 (Sigma Corporation; St. Louis, MO; dilution 1: 1000–1: 2000); mouse anti-α tubulin (Sigma Corporation; St. Louis, MO; dilution 1: 10,000); rabbit anti-GFP (AbCam; Cambridge, MA). Species appropriate secondary antibodies conjugated to horseradish peroxidase (Thermo-Pierce; Waltham, MA) were used at 1: 10,000 and detected using SuperSignal WestDura Western Detection Reagent (Life Technologies; Forest City, CA). The chemiluminescent signal was collected with a Kodak Bioimager 6000 (Carestream Health; Rochester, NY) or SynGene G: Box (SynGene USA; Frederick, MD) and the resulting signal was quantified. Chromatin samples from sub-confluent, confluent, and differentiated CIN612-9E cells were prepared as previously described [62]. Optimal conditions for shearing viral chromatin to 200–500 bp fragments were determined by Southern blot analysis to be similar to that of cellular DNA, and subsequently shearing of total DNA was monitored by agarose gel electrophoresis/ethidium bromide staining. For each cell culture condition, 20 μg chromatin was incubated overnight at 4°C with 1 μg ChromPure Rabbit IgG, whole molecule (011-000-003 (Jackson) ), 2 μg anti-histone H3 antibody (ab1791 (Abcam) ), and 1 μg anti-Sp100 antibody (HPA016707 (Sigma) ). Dynabeads with protein G (Invitrogen) were blocked in 1 mg/ml ultrapure BSA (Ambion) and incubated with samples at 4°C to precipitate chromatin immunocomplexes. Dynabeads were subjected to wash steps and DNA was purified using the ChIP DNA Clean and Concentrator kit (Zymo Research). Genomic regions of immunoprecipitated HPV chromatin were quantified with qPCR using an HPV31 plasmid standard curve. For Sp100 protein quantifications following elution of chromatin-immunocomplexes, 5 μg of the 20 μg IP was processed for ChIP-qPCR and 15 μg processed for immunoblot analysis. For immunoblot analysis, chromatin eluate was analyzed by Sp100 immunoblot analysis, as described above. Chemiluminescent signal was collected using a SynGene G: Box (SynGene USA) and the resulting signal quantified using GeneTools image analysis software (SynGene). To calculate the absolute number of Sp100 molecules in each sample, an Sp100 standard curve was generated by in vitro translation of the pCMV-TnT-EE Sp100A plasmid using the TnT Quick Coupled Transcription/Translation System (Promega). Empty pCMV-TnT-EE vector was translated as a negative control. 35S-methionine (NEN, 1170Ci/mmol) incorporation was determined by TCA precipitation of translated proteins, allowing calculation of moles of Sp100. All images were collected with a Leica SP5 laser scanning confocal microscope (Leica Microsystems) using a 63X oil immersion objective (NA 1. 4). 2D images were collected as a single optical slice for all experiments unless otherwise noted. Images for 3D analysis were collected at either 1 μm or 0. 13 μm thickness per slice, as noted in the figure legends. Where noted, images were deconvolved using Huygens Essential (Scientific Volume Imaging B. V. , VB Hilversum, Netherlands) using manual background selection and a signal-to-noise ratio of 25: 1. Final images were processed using Leica AS AF Lite (Leica Microsystems) or IMARIS (v7. 7. 1; Bitplane AG; Zurich, Switzerland). Exported images were arranged together as a figure, assembled using Adobe Illustrator (Version CS5; Adobe Systems; San Jose, A) and minimally processed as a group using Adobe Photoshop (Version CS5; Adobe Systems; San Jose, CA). Formalin-fixed, paraffin-embedded tissues from normal and HPV16-infected cervix were deparaffinized, rehydrated, and incubated at 96°C for 30 minutes in epitope-retrieval buffer (25 mM Tris-HCl pH 8. 5,1 mM EDTA, 0. 05% SDS) [63]. Sections were blocked with 5% normal donkey serum (NDS) in PBS for one hour in a humidifying chamber at 37°C. Sections were incubated at 37°C with rabbit anti-Sp100 polyclonal antibody (Sigma HPA016707; 1: 500) for one hour in 5% NDS/PBS, washed and incubated at 37°C with goat anti-rabbit polyclonal antibody conjugated to rhodamine red-X (Jackson ImmunoResearch) for one hour in 5% NDS/PBS and washed three times with PBS/0. 05% Tween 20. Sections were incubated at 37°C with anti-E4 antibody conjugated to Alexa 488 (kindly provided by the Doorbar laboratory; 1: 1000) for 1 hour in 5% NDS/PBS, washed three times with PBS/0. 05% Tween 20. Antibodies bound to tissue were briefly fixed with 3: 1 methanol: acetic acid and subsequently 2% paraformaldehyde/PBS. RNA was removed with 1X RNace-IT cocktail (Stratagene) for one hour at 37°C. Sections were dehydrated with an ethanol series and allowed to dry for one hour. 75 ng HPV16 DNA conjugated to Alexa 647 (FISH Tag DNA Multicolor Kit, Invitrogen) in FISH hybridization buffer (Empire Genomics) with 500 ng human Cot-1 DNA (Invitrogen) was applied to each section and sealed with a coverslip. DNA was denatured for 5 minutes at 75°C and hybridized with probe for 20 hours at 37°C with a Thermobrite StatSpin System (Leica). Slides were washed with 1X phosphate buffered detergent (MP Biomedicals), then with 0. 5X SSC with 0. 1% SDS at 65°C, and again with 1X phosphate buffered detergent. Sections were incubated with 300 nM DAPI, washed three times with PBS, and mounted with Prolong Gold (Invitrogen). Images were collected with a Leica Sp5 scanning confocal microscope with a 63X objective (NA 1. 4). Z stacks of individual nuclei were imaged, deconvolved in Huygens Essential (Scientific Volume Imaging B. V. , VB Hilversum, Netherlands) and reconstructed as 3D surfaces in IMARIS (v7. 7. 1; Bitplane AG; Zurich, Switzerland). Primary human keratinocytes were isolated from anonymized neonatal foreskins provided to the Dermatology Branch at NIH from local hospitals. The NIH Institutional Review Board (IRB) approved this process and issued an NIH Institutional Review Board waiver.
Host restriction factors act to limit viral infection as part of the intrinsic immune system. Upon entry into cells, human papillomaviruses (HPV) encounter anti-viral nuclear bodies, called ND10, and components of these bodies, such as Sp100, restrict early viral infection. If HPV escapes these defenses, it can establish a long-term, persistent infection. The eventual production of infectious HPV particles depends on the differentiation program of host keratinocytes. Here we show that in the transition from persistent to productive infection in differentiated keratinocytes, HPV again engages the Sp100 component of ND10 bodies. Sp100 is observed surrounding and inside replication centers in differentiated cells in culture, and in HPV-infected cervical lesions. This results in restriction of viral DNA amplification and late viral gene expression. These data collectively show that Sp100 mediates an anti-viral response at both early and late stages of viral infection.
medicine and health sciences
keratinocytes
pathology and laboratory medicine
gene regulation
pathogens
microbiology
cell differentiation
epithelial cells
viruses
developmental biology
dna replication
dna viruses
hpv-31
dna
epigenetics
chromatin
small interfering rnas
papillomaviruses
chromosome biology
animal cells
medical microbiology
hpv-16
gene expression
microbial pathogens
biological tissue
viral replication
biochemistry
rna
cell biology
nucleic acids
anatomy
virology
viral pathogens
genetics
epithelium
biology and life sciences
cellular types
non-coding rna
organisms
Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle