HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis.

Although most cervical human papillomavirus type 16 (HPV16) infections become undetectable within 1-2 years, persistent HPV16 causes half of all cervical cancers. We used a novel HPV whole-genome sequencing technique to evaluate an exceptionally large collection of 5,570 HPV16-infected case-control samples to determine whether viral genetic variation influences risk of cervical precancer and cancer. We observed thousands of unique HPV16 genomes; very few women shared the identical HPV16 sequence, which should stimulate a careful re-evaluation of the clinical implications of HPV mutation rates, transmission, clearance, and persistence. In case-control analyses, HPV16 in the controls had significantly more amino acid changing variants throughout the genome. Strikingly, E7 was devoid of variants in precancers/cancers compared to higher levels in the controls; we confirmed this in cancers from around the world. Strict conservation of the 98 amino acids of E7, which disrupts Rb function, is critical for HPV16 carcinogenesis, presenting a highly specific target for etiologic and therapeutic research.

A novel HPV16 splicing enhancer critical for viral oncogene expression and cell immortalization.

High-risk carcinogenic human papillomaviruses (HPVs), e.g. HPV16, express the E6 and E7 oncogenes from two mRNAs that are generated in a mutually exclusive manner by splicing. The HPV16 E7 mRNA, also known as the E6*I/E7 mRNA, is produced by splicing between splice sites SD226 and SA409, while E6 mRNAs retain the intron between these splice sites. We show that splicing between HPV16 splice sites SD226 and SA409 is controlled by a splicing enhancer consisting of a perfect repeat of an adenosine-rich, 11 nucleotide sequence: AAAAGCAAAGA. Two nucleotide substitutions in both 11 nucleotide sequences specifically inhibited production of the spliced E6*I/E7 mRNA. As a result, production of E7 protein was reduced and the ability of HPV16 to immortalize human primary keratinocytes was abolished. The splicing-enhancing effect was mediated by the cellular TRAP150/THRAP3 protein that also enhanced splicing of other high-risk HPV E6*I/E7 mRNAs, but had no effect on low-risk HPV mRNAs. In summary, we have identified a novel splicing enhancer in the E6 coding region that is specific for high-risk HPVs and that is critically linked to HPV16 carcinogenic properties.

Diverse tumorigenic consequences of human papillomavirus integration in primary oropharyngeal cancers.

Human papillomavirus (HPV) causes 5% of all cancers and frequently integrates into host chromosomes. The HPV oncoproteins E6 and E7 are necessary but insufficient for cancer formation, indicating that additional secondary genetic events are required. Here, we investigate potential oncogenic impacts of virus integration. Analysis of 105 HPV-positive oropharyngeal cancers by whole-genome sequencing detects virus integration in 77%, revealing five statistically significant sites of recurrent integration near genes that regulate epithelial stem cell maintenance (i.e., SOX2, TP63, FGFR, MYC) and immune evasion (i.e., CD274). Genomic copy number hyperamplification is enriched 16-fold near HPV integrants, and the extent of focal host genomic instability increases with their local density. The frequency of genes expressed at extreme outlier levels is increased 86-fold within ±150 kb of integrants. Across 95% of tumors with integration, host gene transcription is disrupted via intragenic integrants, chimeric transcription, outlier expression, gene breaking, and/or de novo expression of noncoding or imprinted genes. We conclude that virus integration can contribute to carcinogenesis in a large majority of HPV-positive oropharyngeal cancers by inducing extensive disruption of host genome structure and gene expression.

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

The role of HPV11 E7 in modulating STING-dependent interferon ß response in recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare benign tumor caused mainly by the infection of the respiratory tract epithelial cells by the human papillomavirus (HPV) type 6/11. However, the specific mechanisms underlying the inhibition of the host's innate immune response by HPV remain unclear. For this purpose, we employed single-cell RNA sequencing to analyze the states of various immune cells in RRP samples post-HPV infection and utilized a cellular model of HPV infection to elucidate the mechanisms by which HPV evades the innate immune system in RRP. The results revealed distinct immune cell heterogeneity in RRP and demonstrated that HPV11 E7 can inhibit the phosphorylation of the stimulator of interferon genes protein, thereby circumventing the body's antiviral response. In vitro co-culture experiments demonstrated that stimulation of macrophages to produce interferon-beta induced the death of HPV-infected epithelial cells, also reducing HPV viral levels. In summary, our study preliminarily identifies the potential mechanisms by which HPV evades the host's antiviral immune response, as well as the latent antiviral functions exhibited by activated macrophages. This research serves as an initial exploration of antiviral immune evasion in RRP, laying a solid foundation for investigating immunotherapeutic approaches for the disease.IMPORTANCESurgical tumor reduction is the most common treatment for recurrent respiratory papillomatosis (RRP). One of the characteristics of RRP is its persistent recurrence, and multiple surgeries are usually required to control the symptoms. Recently, some adjuvant therapies have shown effectiveness, but none of them can completely clear human papillomavirus (HPV) infection, and thus, a localized antiviral immune response is significant for disease control; after all, HPV infection is limited to the epithelium. Inhibition of interferon-beta (IFN-ß) secretion by HPV11 E7 viral proteins in epithelial cells by affecting stimulator of interferon genes phosphorylation may account for the persistence of low-risk HPV replication in the RRP. Moreover, suppression of the IFN-I pathway in RRP cell types might provide clues regarding the hyporeactive function of local immune cells. However, activation of macrophage groups to produce IFN-ß can still destroy HPV-infected cells.

MmuPV1 E7's interaction with PTPN14 delays Epithelial differentiation and contributes to virus-induced skin disease.

Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7's ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.

BRCA1 deficiency enhances the aggressiveness of breast cancer cells expressing HPV16 oncoproteins.

BACKGROUND INFORMATION: The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. RESULTS: Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-ß and vimentin) were significantly increased in BRCA1-deficient cells. CONCLUSIONS: Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. SIGNIFICANCE: HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.

Evidence for direct interaction between the oncogenic proteins E6 and E7 of high-risk human papillomavirus (HPV).

Human papillomaviruses (HPVs) are DNA tumor viruses that infect mucosal and cutaneous epithelial cells of more than 20 vertebrates. High-risk HPV causes about 5% of human cancers worldwide, and the viral proteins E6 and E7 promote carcinogenesis by interacting with tumor suppressors and interfering with many cellular pathways. As a consequence, they immortalize cells more efficiently in concert than individually. So far, the networks of E6 and E7 with their respective cellular targets have been studied extensively but independently. However, we hypothesized that E6 and E7 might also interact directly with each other in a novel interaction affecting HPV-related carcinogenesis. Here, we report a direct interaction between E6 and E7 proteins from carcinogenic HPV types 16 and 31. We demonstrated this interaction via cellular assays using two orthogonal methods: coimmunoprecipitation and flow cytometry-based FRET assays. Analytical ultracentrifugation of the recombinant proteins revealed that the stoichiometry of the E6/E7 complex involves two E7 molecules and two E6 molecules. In addition, fluorescence polarization showed that (I) E6 binds to E7 with a similar affinity for HPV16 and HPV31 (in the same micromolar range) and (II) that the binding interface involves the unstructured N-terminal region of E7. The direct interaction of these highly conserved papillomaviral oncoproteins may provide a new perspective for studying HPV-associated carcinogenesis and the overall viral life cycle.

Phylogenetic analysis and antigenic epitope prediction for E6 and E7 of Alpha-papillomavirus 9 in Taizhou, China.

BACKGROUND: Alpha-papillomavirus 9 (α-9) is a member of the human papillomavirus (HPV) α genus, causing 75% invasive cervical cancers worldwide. The purpose of this study was to provide data for effective treatment of HPV-induced cervical lesions in Taizhou by analysing the genetic variation and antigenic epitopes of α-9 HPV E6 and E7. METHODS: Cervical exfoliated cells were collected for HPV genotyping. Positive samples of the α-9 HPV single type were selected for E6 and E7 gene sequencing. The obtained nucleotide sequences were translated into amino acid sequences (protein primary structure) using MEGA X, and positive selection sites of the amino acid sequences were evaluated using PAML. The secondary and tertiary structures of the E6 and E7 proteins were predicted using PSIPred, SWISS-MODEL, and PyMol. Potential T/B-cell epitopes were predicted by Industrial Engineering Database (IEDB). RESULTS: From 2012 to 2023, α-9 HPV accounted for 75.0% (7815/10423) of high-risk HPV-positive samples in Taizhou, both alone and in combination with other types. Among these, single-type-positive samples of α-9 HPV were selected, and the entire E6 and E7 genes were sequenced, including 298 HPV16, 149 HPV31, 185 HPV33, 123 HPV35, 325 HPV52, and 199 HPV58 samples. Compared with reference sequences, 34, 12, 10, 2, 17, and 17 nonsynonymous nucleotide mutations were detected in HPV16, 31, 33, 35, 52, and 58, respectively. Among all nonsynonymous nucleotide mutations, 19 positive selection sites were selected, which may have evolutionary significance in rendering α-9 HPV adaptive to its environment. Immunoinformatics predicted 57 potential linear and 59 conformational B-cell epitopes, many of which are also predicted as CTL epitopes. CONCLUSION: The present study provides almost comprehensive data on the genetic variations, phylogenetics, positive selection sites, and antigenic epitopes of α-9 HPV E6 and E7 in Taizhou, China, which will be helpful for local HPV therapeutic vaccine development.

Genetic fusion of CCL11 to antigens enhances antigenicity in nucleic acid vaccines and eradicates tumor mass through optimizing T-cell response.

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer.

Nuclear proinflammatory cytokine S100A9 enhances expression of human papillomavirus oncogenes via transcription factor TEAD1.

Transcription of the human papillomavirus (HPV) oncogenes, E6 and E7, is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed in vitro DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. IMPORTANCE Human papillomavirus (HPV) infection is the primary cause of cervical cancer, and the viral oncogenes E6 and E7 play crucial roles in carcinogenesis. Although cervical inflammation contributes to the development of cervical cancer, the molecular mechanisms underlying the role of these inflammatory responses in HPV carcinogenesis are not fully understood. Our study shows that S100A9, a proinflammatory cytokine, is induced in the nucleus of cervical cancer cells by inflammatory stimuli, and it enhances HPV oncogene expression by acting as a transcriptional coactivator of TEAD1. These findings provide new molecular insights into the relationship between inflammation and viral carcinogenesis.

Human papillomavirus type 16 E6 induces cell competition.

High-risk human papillomavirus (HPV) infections induce squamous epithelial tumors in which the virus replicates. Initially, the virus-infected cells are untransformed, but expand in both number and area at the expense of uninfected squamous epithelial cells. We have developed an in vitro assay in which colonies of post-confluent HPV16 expressing cells outcompete and displace confluent surrounding uninfected keratinocytes. The enhanced colony competition induced by the complete HPV16 genome is conferred by E6 expression alone, not by individual expression of E5 or E7, and requires E6 interaction with p53. E6-expressing keratinocytes undermine and displace adjacent normal keratinocytes from contact with the attachment substrate, thereby expanding the area of the E6-expressing colony at the expense of normal keratinocytes. These new results separate classic oncogenicity that is primarily conferred by HPV16 E7 from cell competition that we show is primarily conferred by E6 and provides a new biological role for E6 oncoproteins from high-risk human papillomaviruses.

Biplex quantitative PCR to detect transcriptionally active human papillomavirus 16 from patient saliva.

Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a RT-qPCR biplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our biplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This biplexing technique allowed for reduced sample input without compromising specificity. In summary, our approach demonstrates the potential to detect viable HPV16 in saliva from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC.

KNTC1, regulated by HPV E7, inhibits cervical carcinogenesis partially through Smad2.

Cervical cancer is the second most common malignancy of the female reproductive tract worldwide. Although cervical cancer is caused by human papillomavirus (HPV) infection, its underlying pathogenesis requires further investigation. The present study investigated the role of kinetochore associated protein 1 (KNTC1) in cervical cancer and its association with the key virus oncoprotein, HPV E7. A series of bioinformatic analyses revealed that KNTC1 might be involved in the tumorigenesis of multiple human malignancies, including cervical cancer. Tissue microarray analysis showed that in vivo KNTC1 expression was higher in high-grade squamous intraepithelial lesions (HSILs) than in normal cervix and even higher in cervical cancer. In vitro silencing of KNTC1 increased the proliferation, invasion and migration of cervical cancer cell lines. Although not affecting apoptosis, KNTC1 silencing significantly promoted G1/S phase transition of the cell cycle. High-throughput analysis of mRNA expression showed that KNTC1 could regulate its downstream target protein Smad2 at the transcriptional level. Moreover, as the key oncoprotein of the virus, HPV E7 could inhibit the expression of KNTC1 protein, and decrease Smad2 protein expression with or without the aid of KNTC1. These results indicated that KNTC1 is a novel tumor suppressor that can impede the initiation and progression of cervical carcinoma, providing insight into the molecular mechanism by which HPV induces cervical cancer.

N6-methyladenosine methylation on FSCN1 mediated by METTL14/IGF2BP3 contributes to human papillomavirus type 16-infected cervical squamous cell carcinoma.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.

hnRNP G/RBMX enhances HPV16 E2 mRNA splicing through a novel splicing enhancer and inhibits production of spliced E7 oncogene mRNAs.

Human papillomavirus type 16 (HPV16) E2 is an essential HPV16 protein. We have investigated how HPV16 E2 expression is regulated and have identifed a splicing enhancer that is required for production of HPV16 E2 mRNAs. This uridine-less splicing enhancer sequence (ACGAGGACGAGGACAAGGA) contains 84% adenosine and guanosine and 16% cytosine and consists of three 'AC(A/G)AGG'-repeats. Mutational inactivation of the splicing enhancer reduced splicing to E2-mRNA specific splice site SA2709 and resulted in increased levels of unspliced E1-encoding mRNAs. The splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G that promoted splicing to SA2709 and enhanced E2 mRNA production. The splicing-enhancing function of hnRNP G mapped to amino acids 236-286 of hnRNP G that were also shown to interact with splicing factor U2AF65. The interactions between hnRNP G and HPV16 E2 mRNAs and U2AF65 increased in response to keratinocyte differentiation as well as by the induction of the DNA damage response (DDR). The DDR reduced sumoylation of hnRNP G and pharmacological inhibition of sumoylation enhanced HPV16 E2 mRNA splicing and interactions between hnRNP G and E2 mRNAs and U2AF65. Intriguingly, hnRNP G also promoted intron retention of the HPV16 E6 coding region thereby inhibiting production of spliced E7 oncogene mRNAs.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Detection of Human Papillomavirus DNA, E6/E7 Messenger RNA, and p16INK4a in Lung Cancer: A Systematic Review and Meta-analysis.

BACKGROUND: The etiologic link between human papillomavirus (HPV) and lung cancer is still controversial. METHODS: PubMed and Cochrane databases were searched from inception to December 2020 to identify studies on the infection of HPV in lung cancer. We calculated the attributable proportion of HPV in lung cancer by pooling the infection of cases positive for both HPV DNA and biomarkers of carcinogenesis that may be induced by HPV (E6/E7 messenger RNA or p16INK4a). RESULTS: A total of 117 studies, comprising data of 12 616 lung cancer cases from 22 countries across 5 continents, were included. The overall HPV DNA positivity in primary lung cancer cases worldwide was 16.4% (95% confidence interval, 12.7%-20.5%). HPV DNA positivity of lung cancer varied significantly by pathological type and geographic region. Notably, the expression rate of p16INK4a is significantly higher than the positivity of HPV DNA and of HPV E6/E7 mRNA (P < .05). The estimate of HPV attributable proportion defined by expression of E6/E7 mRNA was 0 and of p16INK4a was 7.3%. CONCLUSIONS: The data in this systematic review is robust enough to contradict the possible participation of HPV in lung cancer carcinogenesis. Prophylactic vaccines targeting HPV cannot have the potential to prevent lung cancer.

The role of RNA-binding proteins in the processing of mRNAs produced by carcinogenic papillomaviruses.

Human papillomaviruses (HPV) are epitheliotropic DNA tumor viruses that are prevalent in the human population. A subset of the HPVs termed high-risk HPVs (HR-HPVs) are causative agents of anogenital cancers and head-and-neck cancers. Cancer is the result of persistent high-risk HPV infections that have not been cleared by the immune system of the host. These infections are characterized by dysregulated HPV gene expression, in particular constitutive high expression of the HPV E6 and E7 oncogenes and absence of the highly immunogenic viral L1 and L2 capsid proteins. HPVs make extensive use of alternative mRNA splicing to express its genes and are therefore highly dependent on cellular RNA-binding proteins for proper gene expression. Levels of RNA-binding proteins are altered in HPV-containing premalignant cervical lesions and in cervical cancer. Here we review our current knowledge of RNA-binding proteins that control HPV gene expression. We focus on RNA-binding proteins that control expression of the E6 and E7 oncogenes since they initiate and drive development of cancer and on the immunogenic L1 and L2 proteins as there silencing may contribute to immune evasion during carcinogenesis. Furthermore, cellular RNA-binding proteins are essential for HPV gene expression and as such may be targets for therapy to HPV infections and HPV-driven cancers.

The HPV16 E6, E7/miR-23b-3p/ICAT signaling axis promotes proliferation, migration, invasion and EMT of cervical cancer cells.

Cervical cancer (CC) remains one of the most common female malignancies, with higher incidence and mortality rates. more than 99% of CCs are associated with persistent infection with high-risk human papillomavirus. In view of the growing evidence that HPV 16 E6 and E7, two key oncoproteins encoded by HPV 16, regulate the expression of many other multifunctional genes and downstream effectors that contribute to the development of CC. Herein, we undertook a comprehensive effort into how HPV16 E6, E7 oncogenes affect the progression of CC cells. Previous studies have shown that ICAT expression was significantly increased in CC and had a pro-cancer effect. We observed that knockdown of HPV16 E6, E7 expression in SiHa and CasKi cells resulted in significant inhibition of ICAT expression and upregulation of miR-23b-3p expression. Besides, dual luciferase assays confirmed that ICAT was a target gene of miR-23b-3p, and negatively modulated by miR-23b-3p. Functional experiments showed that the overexpression of miR-23b-3p suppressed malignant behaviors of CC cells, such as migration, invasion and EMT. The overexpression of ICAT counteracted the suppressive effect of miR-23b-3p on HPV16-positive CC cells. Furthermore, after the knockdown of HPV16 E6 and E7, the inhibition of miR-23b-3p could increase the ICAT expression and rescue the siRNA HPV16 E6, E7-mediated suppressive impact on the aggressiveness of SiHa and CaSki cells. Collectively, our findings uncover that HPV16 E6, E7/miR-23b-3p/ ICAT axis plays an important role in HPV16-positive CC pathogenesis, which may serve as a promising therapeutic target for HPV16-associated CC.