High-risk human papillomaviruses repress constitutive kappa interferon transcription via E6 to prevent pathogen recognition receptor and antiviral-gene expression.
ABSTRACT: 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:Keratinocyte infection with high-risk human papillomavirus genotypes has been linked to cancer development. In cervix, the alpha HPV16 and HPV18 have been reported as the mayor causative agents of cervical cancer. Oncogenic progression and chronic inflammation are closely related processes, with IL-6 as one of the main pro-inflammatory cytokines involved. However, there are limited studies about the regulation of IL-6 by low and high risk HPVs and the HPV proteins implicated in this modulation. In this work, we report the overexpression of IL-6 in HPV infected cervical cancer derived cell lines (HeLa and SiHa) compared to non-tumorigenic keratinocytes (HaCaT), and in Cervical Intraepithelial Neoplasia grade 1 HPV16 and HPV18 positive cervical samples compared to HPV negative samples without lesions. Moreover, we generated HaCaT keratinocytes that express E5, E6, and E7 from high risk (16 or 18) or low risk (62 and 84) HPVs. E5 proteins do not modify IL-6 expression, while E7 modestly increase it. Interestingly, E6 proteins in HaCaT cells upregulate IL-6 mRNA expression and protein secretion. Indeed, in HaCaT cells that express high risk HPV16E6 or HPV18E6 proteins, only the truncated E6<sup>*</sup> isoforms were expressed, showing the stronger IL-6 overexpression, while in HaCaT cells that express low risk HPV62 and HPV84 full length E6 proteins, IL-6 was also upregulated but not so drastically. Since HaCaT cells have a mutated p53 form that is not degraded by the introduction of E6 or E6/E7, it seems that E6/E7 regulate IL-6 by an additional mechanism independent of p53. In addition, basal keratinocytes showed a strong expression of IL-6R using immunohistochemistry, suggesting an autocrine mechanism over proliferative cells. Altogether, IL-6 cytokine expression in keratinocytes is upregulated by E6 and E7 proteins from HPVs 16, 18, 62, and 84, especially by high risk HPV16 and HPV18 E6<sup>*</sup>, which may contribute to promote a pro-inflammatory and highly proliferative microenvironment that can persist over time and lead to cervical tumorigenesis.
Project description:Human papillomaviruses (HPVs) infect keratinocytes of stratified epithelia. Long-term persistence of infection is a critical risk factor for the development of HPV-induced malignancies. Through the actions of its oncogenes, HPV evades host immune responses to facilitate its productive life cycle. In this work, we discovered a previously unknown function of the HPV16 E5 oncoprotein in the suppression of interferon (IFN) responses. This suppression is focused on keratinocyte-specific IFN-? and is mediated through E5-induced changes in growth factor signaling pathways, as identified through phosphoproteomics analysis. The loss of E5 in keratinocytes maintaining the complete HPV16 genome results in the derepression of IFNK transcription and subsequent JAK/STAT-dependent upregulation of several IFN-stimulated genes (ISGs) at both the mRNA and protein levels. We also established a link between the loss of E5 and the subsequent loss of genome maintenance and stability, resulting in increased genome integration.IMPORTANCE Persistent human papillomavirus infections can cause a variety of significant cancers. The ability of HPV to persist depends on evasion of the host immune system. In this study, we show that the HPV16 E5 protein can suppress an important aspect of the host immune response. In addition, we find that the E5 protein is important for helping the virus avoid integration into the host genome, which is a frequent step along the pathway to cancer development.
Project description:The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16INK4a accumulation, whereas no p16INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies.IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.
Project description:In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-? that is involved in immune evasion and HPV persistence. To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-? and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-? and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-? were further determined in clinical specimens by real time PCR. STING and IFN-? were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-? transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.
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.
Project description:The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation.<h4>Importance</h4>High-risk human papillomaviruses (HPVs) are the causative agents of almost all cervical cancers and many other cancers, including anal, vaginal, vulvar, penile, and oropharyngeal cancers. Despite the availability of efficacious HPV vaccines, it is critical to determine how HPVs cause cancer, as many people remain unvaccinated and the vaccine does not prevent cancer development in individuals who are already infected. Two HPV proteins, E6 and E7, are the major drivers of cancer development, and much remains to be learned about how the expression of these viral proteins reprograms infected cells, ultimately resulting in cancer development. Small, noncoding human RNAs, termed microRNAs (miRs), regulate gene expression and have been implicated in almost all human cancers, including HPV-associated cancers. Our study provides a comprehensive analysis of how E6 and E7 alter the expression of human miRs and how this potentially impacts cellular gene expression, which may contribute to cancer development.
Project description:The Notch signaling pathway is a key determinant in keratinocyte differentiation and growth cycle arrest, and has been reported to have a tumor suppressor function in skin. The papillomavirus life cycle is intricately linked to the differentiation status of keratinocytes. Papillomaviruses are associated with benign proliferative epithelial lesions in their respective hosts. Although human papillomaviruses (HPVs) associated with genital tract lesions have been extensively studied, studies of the cutaneous HPVs are more limited. In particular, it is well established that the E6 proteins of high-risk HPVs of the ?-genus such as HPV16 and HPV18 mediate the degradation of p53 by its association with the ubiquitin ligase E6AP. In contrast, less is known about the cellular activities of the cutaneous HPVs of the ?-genus. By using an unbiased proteomic approach, we identify MAML1 and other members of the Notch transcription complex as high-confidence cellular interacting proteins of E6 proteins of the ?-genus HPVs and of the bovine papillomavirus type 1 associated with cutaneous fibropapillomas. We show that bovine papillomavirus type 1 and ?-HPV E6 repress Notch transcriptional activation, and that this repression is dependent on an interaction with MAML1. Finally, we show that the expression levels of endogenous Notch target genes are repressed by ?-HPV E6 proteins. These findings elucidate a mechanism of viral antagonism of Notch signaling, and suggest that Notch signaling is an important epithelial cell pathway target for the ?-HPVs.
Project description:Background:Persistent infection with human papillomaviruses (HPVs) has been associated with cervical intraepithelial neoplasia (CIN) and cervical cancer. However, why only a fraction of HPV cases progress to cancer is still unclear. Methods:We focused on the heterogeneity, classification, evolution and dispersal of variants for 14 common HPV types in 262 HPV-positive patients with cervical lesions. The E6 and E7 genes of HPV were sequenced and compared with the HPV reference for sequence analysis. Phylogenetic trees were constructed using the neighbour-joining tree method with MEGA 7.0. Results:In this study, 233 E6 and 212 E7 sequences were successfully amplified by PCR, and these sequences were divided into 5 species groups: alpha-9 (HPV16, 31, 33, 52, 58), alpha-5 (HPV51), alpha-6 (HPV53, 66), alpha-7 (HPV18, 39, 59, 68) and alpha-10 (HPV6, 44). The incidence of high-grade squamous intraepithelial lesion (HSIL) in patients infected with alpha-9 HPV was significantly increased compared with other groups (P <?0.0001), especially HPV16 (P <?0.0001). Strikingly, E7 had significantly fewer nonsynonymous variants in the HSIL compared to <HSIL groups (P =?3.17× 10-?4). The A388C (K93?N) variation in HPV58 E6 can significantly reduce the risk of HSIL (P =?0.015). However, T7220G (D32E) variation in HPV16 E6 and A7689G (N29S) in HPV16 E7 increased the incidence of HSIL compared to the <HSIL group (P =?0.036 and 0.022). Conclusions:Strict conservation of E7 is important for HPV carcinogenicity, especially N29 of HPV16. The findings in this work provide preventative/therapeutic interventions for HPV infections and CIN.
Project description:Certain types of Human papilloma viruses (HPV) are the etiological agents for cervical cancer. However, not all infections of high-risk HPVs will finally lead to cancer since most HPV infections are cleared without any consequences. Chlamydia trachomatis is the most prevalent sexual transmitted bacteria and is an obligatory intracellular pathogen exhibiting tropism in endocervical epithelial cells. Over the past decades, C. trachomatis is thought to be a potential co-factor for cervical cancer formation, but there are also studies that did not show such a correlation. To address this question in molecular terms, we stably expressed HPV16 E6 and E7 in spontaneously immortalized NOKs (normal oral keratinocytes) and performed SILAC (stable isotope labeling by amino acids in cell culture) with or without C. trachomatis infection to study the impact of HPV16 oncogene expression and C. trachomatis infection on host proteome changes.
Project description:Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs) and HFKs containing either wild-type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Interferon (IFN)-stimulated genes (ISGs) and extracellular matrix remodeling genes were suppressed, the most prominent pathways affected. STAT1, IFNAR1, IRF3, and IRF7 were knocked down in stromal fibroblasts using lentiviral short hairpin RNA (shRNA) transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated with increased late keratinocyte differentiation but not decreased IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation.IMPORTANCE The persistence of high-risk human papillomavirus (HPV) infections is the key risk factor for developing HPV-associated cancers. The ability of HPV to evade host immunity is a critical component of its ability to persist. The environment surrounding a tumor is increasingly understood to be critical in cancer development, including immune evasion. Our studies show that HPV can suppress the expression of immune-related genes in neighboring fibroblasts in a three-dimensional (3D) model of human epithelium. This finding is significant, because it indicates that HPV can control innate immunity not only in the infected cell but also in the microenvironment. In addition, the ability of HPV to regulate stromal gene expression depends in part on the viral oncogene E5, revealing a new function for this protein as an immune evasion factor.