Post-Transcriptional Regulation of KLF4 by High-Risk Human Papillomaviruses Is Necessary for the Differentiation-Dependent Viral Life Cycle.
ABSTRACT: Human papillomaviruses (HPVs) are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. A subset of the over 200 HPV types, referred to as high-risk, are the causative agents of most anogenital malignancies. HPVs infect cells in the basal layer, but restrict viral genome amplification, late gene expression, and capsid assembly to highly differentiated cells that are active in the cell cycle. In this study, we demonstrate that HPV proteins regulate the expression and activities of a critical cellular transcription factor, KLF4, through post-transcriptional and post-translational mechanisms. Our studies show that KLF4 regulates differentiation as well as cell cycle progression, and binds to sequences in the upstream regulatory region (URR) to regulate viral transcription in cooperation with Blimp1. KLF4 levels are increased in HPV-positive cells through a post-transcriptional mechanism involving E7-mediated suppression of cellular miR-145, as well as at the post-translational level by E6-directed inhibition of its sumoylation and phosphorylation. The alterations in KLF4 levels and functions results in activation and suppression of a subset of KLF4 target genes, including TCHHL1, VIM, ACTN1, and POT1, that is distinct from that seen in normal keratinocytes. Knockdown of KLF4 with shRNAs in cells that maintain HPV episomes blocked genome amplification and abolished late gene expression upon differentiation. While KLF4 is indispensable for the proliferation and differentiation of normal keratinocytes, it is necessary only for differentiation-associated functions of HPV-positive keratinocytes. Increases in KLF4 levels alone do not appear to be sufficient to explain the effects on proliferation and differentiation of HPV-positive cells indicating that additional modifications are important. KLF4 has also been shown to be a critical regulator of lytic Epstein Barr virus (EBV) replication underscoring the importance of this cellular transcription factor in the life cycles of multiple human cancer viruses.
Project description:Infections by low-risk papillomavirus types, such as human papillomavirus (HPV) type 6 (HPV-6) and HPV-11, induce benign genital warts that rarely progress to malignancy. In contrast, lesions induced by high-risk HPV types have the potential to progress to cancer. Considerable information is available concerning the pathogenesis of high-risk HPV types, but little is known about the life cycle of low-risk HPV types. Although functionally distinct, both high- and low-risk virus types infect keratinocytes and induce virion production upon differentiation. This information suggests that they may share common mechanisms for regulating their productive life cycles. Using tissue culture methods developed to study high-risk HPV types, we examined the ability of HPV-11 to be stably maintained as episomes following transfection of normal human keratinocytes with cloned viral DNA. HPV-11 genomes were found to be maintained in keratinocytes for extended passages in cultures in 14 independent experiments involving transfection of cloned HPV-11 DNA. Interestingly, the HPV-11-positive cells exhibited an extended life span that averaged approximately twofold longer than that of control neomycin-transfected cells. In organotypic cultures, HPV-11-positive cells exhibited altered differentiation patterns, but the extent of disruption was less severe than that seen with high-risk HPV types. In addition, the amplification of HPV-11 DNA, as well as the induction of several viral messages, was observed following differentiation of transfected cells in semisolid media. To determine whether global changes in cellular gene expression induced by HPV-11 were similar to those observed with high-risk HPV-31 (Y. E. Chang and L. A. Laimins, J. Virol. 74:4174-4182, 2000), microarray analysis of 7,075 expressed sequences was performed. A spectrum of cellular genes different from that previously reported for HPV-31 was found to be activated or repressed by HPV-11. The expression of only a small set of genes was similarly altered by both high- and low-risk HPV types. This result suggests that different classes of HPVs have distinct effects on global cellular transcription patterns during infection. The methods described allow for a genetic analysis of HPV-11 in the context of its differentiation-dependent life cycle.
Project description:Methylation of the high-risk human papillomavirus type 16 (HPV16) upstream regulatory region (URR) has been described, but whether methylation is present among low-risk HPVs is unknown. The methylation status of the HPV6 URR was analyzed in papillomas from the upper aerodigestive tract of six adult patients. All CpGs in the URR were unmethylated, from both basal/intermediate and superficial cells, suggesting that methylation is not involved in the regulation of transcription from the HPV6 URR, regardless of epithelial differentiation.
Project description:Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have potentially important implications for HPV control.
Project description:High-risk human papillomaviruses (HPVs) constitutively activate the ataxia telangiectasia and Rad3-related (ATR) DNA damage response pathway, and this is required for viral replication. In fibroblasts, activated ATR regulates transcription of inflammatory genes through its negative effects on the autophagosome cargo protein p62. In addition, suppression of p62 results in increased levels of the transcription factor GATA4, leading to cellular senescence. In contrast, in HPV-positive keratinocytes, we observed that activation of ATR resulted in increased levels of phosphorylated p62, which in turn lead to reduced levels of GATA4. Knockdown of ATR in HPV-positive cells resulted in decreased p62 phosphorylation and increased GATA4 levels. Transcriptome sequencing (RNA-seq) analysis of HPV-positive cells identified inflammatory genes and interferon factors as negative transcriptional targets of ATR. Furthermore, knockdown of p62 or overexpression of GATA4 in HPV-positive cells leads to inhibition of viral replication. These findings identify a novel role of the ATR/p62 signaling pathway in HPV-positive cells.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate the ATR DNA damage repair pathway, and this is required for HPV genome amplification. In the present study, we show that HPV-induced ATR activation also leads to suppression of expression of inflammatory response genes. This suppression results from HPV-induced phosphorylation of the autophagosome cargo protein p62 which regulates the levels of the transcription factor GATA4. Activation of p62 in normal fibroblasts results in senescence, but this is not seen in HPV-positive keratinocytes. Importantly, knockdown of p62 or overexpression of GATA4 in HPV-positive cells abrogates viral replication. This study demonstrates that activation of ATR in HPV-positive cells triggers a p62-directed pathway inducing suppression of inflammatory gene expression independent of DNA repair and facilitating HPV replication.
Project description:High risk human papillomaviruses are squamous epitheliotropic viruses that may cause cervical and other cancers. HPV replication depends on squamous epithelial differentiation. Transformation of HPV-infected cells goes along with substantial alteration of the viral gene expression profile and preferentially occurs at transformation zones usually at the uterine cervix. Methylation of the viral genome may affect regulatory features that control transcription and replication of the viral genome. Therefore, we analyzed the methylation pattern of the HPV16 upstream regulatory region (URR) during squamous epithelial differentiation and neoplastic transformation and analyzed how shifts in the HPV URR methylome may affect viral gene expression and replication. HPV 16 positive biopsy sections encompassing all stages of an HPV infection (latent, permissive and transforming) were micro-dissected and DNA was isolated from cell fractions representing the basal, intermediate, and superficial cell layers, each, as well as from transformed p16(INK4a)-positive cells. We observed fundamental changes in the methylation profile of transcription factor binding sites in the HPV16 upstream regulatory region linked to the squamous epithelial differentiation stage. Squamous epithelial transformation indicated by p16(INK4a) overexpression was associated with methylation of the distal E2 binding site 1 leading to hyper-activation of the HPV 16 URR. Adjacent normal but HPV 16-infected epithelial areas retained hyper-methylated HPV DNA suggesting that these viral genomes were inactivated. These data suggest that distinct shifts of the HPV 16 methylome are linked to differentiation dependent transcription and replication control and may trigger neoplastic transformation.
Project description:Human papillomaviruses (HPV) are the causative agents of cervical cancers. The infectious HPV life cycle is closely linked to the differentiation state of the host epithelia, with viral genome amplification, late gene expression and virion production restricted to suprabasal cells. The E6 and E7 proteins provide an environment conducive to DNA synthesis upon differentiation, but little is known concerning the mechanisms that regulate productive viral genome amplification. Using keratinocytes that stably maintain HPV-31 episomes, and chemical inhibitors, we demonstrate that viral proteins activate the ATM DNA damage response in differentiating cells, as indicated by phosphorylation of CHK2, BRCA1 and NBS1. This activation is necessary for viral genome amplification, as well as for formation of viral replication foci. In contrast, inhibition of ATM kinase activity in undifferentiated keratinocytes had no effect on the stable maintenance of viral genomes. Previous studies have shown that HPVs induce low levels of caspase 3/7 activation upon differentiation and that this is important for cleavage of the E1 replication protein and genome amplification. Our studies demonstrate that caspase cleavage is induced upon differentiation of HPV positive cells through the action of the DNA damage protein kinase CHK2, which may be activated as a result of E7 binding to the ATM kinase. These findings identify a major regulatory mechanism responsible for productive HPV replication in differentiating cells. Our results have potential implications for the development of anti-viral therapies to treat HPV infections.
Project description:Human papillomaviruses (HPV) regulate their differentiation-dependent life cycles by activating a number of cellular pathways, such as the DNA damage response, through control of post-translational protein modification. Sirtuin 1 (SIRT1) is a protein deacetylase that modulates the acetylation of a number of cellular substrates, resulting in activation of pathways controlling gene expression and DNA damage repair. Our studies indicate that SIRT1 levels are increased in cells containing episomes of high-risk HPV types through the combined action of the E6 and E7 oncoproteins. Knockdown of SIRT1 in these cells with shRNAs impairs viral activities including genome maintenance, amplification and late gene transcription, with minimal effects on cellular proliferation ability. Abrogation of amplification was also seen following treatment with the SIRT1 deacetylase inhibitor, EX-527. Importantly, SIRT1 binds multiple regions of the HPV genome in undifferentiated cells, but this association is lost upon of differentiation. SIRT1 regulates the acetylation of Histone H1 (Lys26) and H4 (Lys16) bound to HPV genomes and this may contribute to regulation of viral replication and gene expression. The differentiation-dependent replication of high-risk HPVs requires activation of factors in the Ataxia Telangiectasia Mutated (ATM) pathway and SIRT1 regulates the recruitment of both NBS1 and Rad51 to the viral genomes. These observations demonstrate that SIRT1 is a critical regulator of multiple aspects of the high-risk HPV life cycle.
Project description:Persistent infections with human papillomavirus type 16 (HPV16), HPV18, or HPV31 are necessary for the development of cervical cancer, implying that HPVs have evolved immunoevasive mechanisms. Recent global transcriptome analyses indicated that these HPV types downregulate the constitutive expression of interferon (IFN)-stimulated genes (ISGs), but the underlying mechanism is not well understood. Comparative analyses of ISG transcription in keratinocytes with complete HPV16, -18, and -31 genomes revealed that antiviral genes (IFIT1 and MX1), genes involved in IFN signaling (STAT1), proapoptotic genes (TRAIL and XAF1), and pathogen recognition receptors (TLR3, RIG-I, and MDA5) are inhibited to similar extents by HPV16, -18, and -31. The lower expression of pathogen receptors in HPV-positive cells correlated with a greatly impaired induction of IFN-? and also of IFN-?1, -2, and -3 upon receptor stimulation. IFN-? is constitutively expressed in normal keratinocytes and is strongly repressed by HPV16, -18, and -31. ISGs downregulated in HPV-positive cells can be reactivated by IFN-? expression. The viral E6 and E7 oncogenes are sufficient for IFN-? repression, with E6 being mainly responsible. E6 inhibits IFN-? transcription independently from binding to PDZ proteins. IFN-? expression can be activated in only one cell line by E6AP knockdown but can be activated in all tested HPV-positive cells by addition of a DNA methyltransferase inhibitor, suggesting that HPVs modulate DNA methylation. Taken together, these results suggest that carcinogenic HPVs target IFN-? by different pathways in keratinocytes to inhibit both antiviral ISGs and pathogen recognition receptors, which in turn reduces the expression of inducible IFNs.
Project description:The life cycle of human papillomaviruses (HPVs) is linked to epithelial differentiation, with late viral events restricted to the uppermost stratified layers. Our studies indicated that HPV activates capases-3, -7, and -9 upon differentiation, whereas minimal activation was observed in differentiating normal keratinocytes. Activation occurred in the absence of significant levels of apoptosis, suggesting a potential role for caspases in the viral life cycle. In support of this, the addition of caspase inhibitors significantly impaired differentiation-dependent viral genome amplification. A conserved caspase cleavage motif was identified in the replication protein E1 ((46)DxxD(49)) that was targeted in vitro by both recombinant caspase-3 and caspase-7. Mutation of this site inhibited amplification of viral genomes, indicating that caspase cleavage is necessary for the productive viral life cycle. Our study demonstrates that HPV activates caspases upon differentiation to facilitate productive viral replication and represents a way by which HPV controls viral gene function in differentiating cells.
Project description:Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1?) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1? production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1? processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1? regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1? was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1? that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1? is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1? levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1? regulation which ultimately inhibits the secretion of IL-1? in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1? towards cervical cancer could be discerned. Hence, attenuation of IL-1? by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.