Engineering of Recombinant Human Papillomavirus 16 L1 Protein for Incorporation with para-Azido-L-Phenylalanine.

Human papillomavirus (HPV) L1 capsid protein were produced in several host systems, but few studies have focused on enhancing the properties of the L1 protein. In this study, we aimed to produce recombinant Human papillomavirus (HPV) L1 capsid protein containing para-azido-L-phenylalanine (pAzF) in Escherichia coli. First, we expressed the maltose-binding protein (MBP)-fused HPV16 L1, and 5 residues in HPV16 L1 protein were selected by the in silico modeling for amber codon substitution. Among the variants of the five locations, we identified a candidate that exhibited significant differences in expression with and without pAzF via genetic code expansion (GCE). The expressed recombinant MBP-HPV16L1 protein was confirmed for incorporation of pAzF and the formation of VLPs was tested in vitro.

Manipulating host secreted protein gene expression: an indirect approach by HPV11/16 E6/E7 to suppress PBMC cytokine secretion.

Human papillomavirus (HPV) 11/16 E6/E7 proteins have been recognized to be pivotal in viral pathogenesis. This study sought to uncover the potential mechanisms of how HPV11/16 E6/E7-transfected keratinocytes inhibit cytokine secretion in peripheral blood mononuclear cells (PBMC). Upon co-culturing HPV11/16 E6/E7-transfected keratinocytes with PBMC in a non-contact manner, we observed a marked decrease in various cytokines secreted by PBMC. To determine if this suppression was mediated by specific common secreted factors, we conducted transcriptomic sequencing on these transfected cells. This analysis identified 53 common differentially secreted genes in all four HPV-transfected cells. Bioinformatics analysis demonstrated these genes were predominantly involved in immune regulation. Results from quantitative PCR (qPCR) and an extensive literature review suggested the downregulation of 12 genes (ACE2, BMP3, BPIFB1, CLU, CST6, CTF1, HMGB2, MMP12, PDGFA, RNASE7, SULF2, TGM2), and upregulation of 7 genes (CCL17, CCL22, FBLN1, PLAU, S100A7, S100A8, S100A9), may be crucial in modulating tumor immunity and combating pathogenic infections, with genes S100A8 and S100A9, and IL-17 signaling pathway being particularly noteworthy. Thus, HPV11/16 E6/E7 proteins may inhibit cytokine secretion of immune cells by altering the expression of host-secreted genes. Further exploration of these genes may yield new insights into the complex dynamics of HPV infection.

Translational control of papillomavirus mRNAs in the spotlight.

High-risk human papillomaviruses (HPVs) cause most cases of cervical cancer, a disease with an increasing impact worldwide. Recent studies have shown that the synthesis of viral oncoproteins is strongly subject to translational control. Thus, targeting the protein synthesis machinery might open novel avenues to develop innovative therapies aiming to improve patients' survival.

Tirbanibulin decreases cell proliferation and downregulates protein expression of oncogenic pathways in human papillomavirus containing HeLa cells.

Tirbanibulin 1% ointment is a synthetic antiproliferative agent approved in 2021 by the European Union for treating actinic keratoses (AK). Topical tirbanibulin has clinically resolved HPV-57 ( +) squamous cell carcinoma (SCC), HPV-16 ( +) vulvar high-grade squamous intraepithelial lesion, epidermodysplasia verruciformis, and condyloma. We examined how tirbanibulin might affect HPV oncoprotein expression and affect other cellular pathways involved in cell proliferation and transformation. We treated the HeLa cell line, containing integrated HPV-18, with increasing doses of tirbanibulin to determine the effects on cell proliferation. Immunoblotting was performed with antibodies against the Src canonical pathway, HPV 18 E6 and E7 transcription regulation, apoptosis, and invasion and metastasis pathways. Cell proliferation assays with tirbanibulin determined the half-maximal inhibitory concentration (IC50) of HeLa cells to be 31.49 nmol/L. Increasing concentrations of tirbanibulin downregulates the protein expression of Src (p < 0.001), phospho-Src (p < 0.001), Ras (p < 0.01), c-Raf (p < 0.001), ERK1 (p < 0.001), phospho-ERK1 (p < 0.001), phospho-ERK2 (p < 0.01), phospho-Mnk1 (p < 0.001), eIF4E (p < 0.01), phospho-eIF4E (p < 0.001), E6 (p < 0.01), E7 (p < 0.01), Rb (p < 0.01), phospho-Rb (p < 0.001), MDM2 (p < 0.01), E2F1 (p < 0.001), phospho-FAK (p < 0.001), phospho-p130 Cas (p < 0.001), Mcl-1 (p < 0.01), and Bcl-2 (p < 0.001), but upregulates cPARP (p < 0.001), and cPARP/fPARP (p < 0.001). These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins via the Src- MEK- pathway. Tirbanibulin significantly downregulates oncogenic proteins related to cell cycle regulation and cell proliferation while upregulating apoptosis pathways.

Tirbanibulin is Promising Novel Therapy for Human Papillomavirus (HPV)-associated Diseases.Tirbanibulin 1% ointment is an approved synthetic topical ointment for treating actinic keratoses (AK), a precancer of skin cancer. Topical tirbanibulin has previously been reported to clinically resolve human papillomavirus (HPV)-( +) diseases.In this study, we examine how tirbanibulin may affect the HPV and pathways associated with cancer.We treated the HeLa cell line to determine the effects on HPV cell proliferation. Increasing the concentration of tirbanibulin statistically significantly affected numerous cellular pathways often associated with cancer.These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins and thereby kill cancer cells.

Senataxin mediates R-loop resolution on HPV episomes.

Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.

In-silico study unveils potential phytocompounds in Andrographis paniculata against E6 protein of the high-risk HPV-16 subtype for cervical cancer therapy.

Despite therapeutic advancements, cervical cancer caused by high-risk subtypes of the human papillomavirus (HPV) remains a leading cause of cancer-related deaths among women worldwide. This study aimed to discover potential drug candidates from the Asian medicinal plant Andrographis paniculata, demonstrating efficacy against the E6 protein of high-risk HPV-16 subtype through an in-silico computational approach. The 3D structures of 32 compounds (selected from 42) derived from A. paniculata, exhibiting higher binding affinity, were obtained from the PubChem database. These structures underwent subsequent analysis and screening based on criteria including binding energy, molecular docking, drug likeness and toxicity prediction using computational techniques. Considering the spectrometry, pharmacokinetic properties, docking results, drug likeliness, and toxicological effects, five compounds-stigmasterol, 1H-Indole-3-carboxylic acid, 5-methoxy-, methyl ester (AP7), andrographolide, apigenin and wogonin-were selected as the potential inhibitors against the E6 protein of HPV-16. We also performed 200 ns molecular dynamics simulations of the compounds to analyze their stability and interactions as protein-ligand complexes using imiquimod (CID-57469) as a control. Screened compounds showed favorable characteristics, including stable root mean square deviation values, minimal root mean square fluctuations and consistent radius of gyration values. Intermolecular interactions, such as hydrogen bonds and hydrophobic contacts, were sustained throughout the simulations. The compounds displayed potential affinity, as indicated by negative binding free energy values. Overall, findings of this study suggest that the selected compounds have the potential to act as inhibitors against the E6 protein of HPV-16, offering promising prospects for the treatment and management of CC.

The BICD2 dynein cargo adaptor binds to the HPV16 L2 capsid protein and promotes HPV infection.

During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.

HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes, including viruses, such as HPV, which must infect and manipulate skin keratinocyte stem cells (KSCs) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induced ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses. Together, these results define the "hit-and-run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lack melanosome protection and are thus susceptible to sun light-induced malignant transformation.

Small molecule NMD and MDM2 inhibitors synergistically trigger apoptosis in HeLa cells.

The nonsense-mediated mRNA decay (NMD) pathway and the p53 pathway, linked to tumorgenesis, are also promising targets for cancer treatment. NMD plays an important role in RNA quality control, while the p53 pathway is involved in cancer suppression. However, their individual and combined effects on cervical cancer are poorly understood. In this study, we evaluated the impacts of NMD inhibitor, Mouse double minute 2 homolog (MDM2) inhibitor, and their combination on cell apoptosis, cell cycle, and p53 target genes in human papillomavirus-18-positive HeLa cells. Our findings revealed that XR-2 failed to activate p53 or induce apoptosis in HeLa cells, whereas SMG1 (serine/threonine-protein kinase 1) inhibitor repressed cell proliferation at high concentrations. Notably, the combination of these 2 agents significantly inhibited cell proliferation, arrested the cell cycle, and triggered cell apoptosis. Mechanistically, MDM2 inhibitor and NMD inhibitor likely exert a synergistically through the truncated E6 protein. These results underscore the potential of employing a combination of MDM2 inhibitor and NMD inhibitor as a promising candidate for the clinical treatment of human papillomavirus-infected tumors.

Understanding the HPV associated cancers: A comprehensive review.

Human papillomavirus (HPV), a common cause of sexually transmitted diseases, may cause warts and lead to various types of cancers, which makes it important to understand the risk factors associated with it. HPV is the leading risk factor and plays a crucial role in the progression of cervical cancer. Viral oncoproteins E6 and E7 play a pivotal role in this process. Beyond cervical cancer, HPV-associated cancers of the mouth and throat are also increasing. HPV can also contribute to other malignancies like penile, vulvar, and vaginal cancers. Emerging evidence links HPV to these cancers. Research on the oncogenic effect of HPV is still ongoing and explorations of screening techniques, vaccination, immunotherapy and targeted therapeutics are all in progress. The present review offers valuable insight into the current understanding of the role of HPV in cancer and its potential implications for treatment and prevention in the future.

HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

Oncogenic HPV E6 proteins have a PDZ-binding motif (PBM) which plays important roles in both the viral life cycle and tumor development. The PBM confers interaction with a large number of different PDZ domain-containing substrates, one of which is Sorting Nexin 27. This protein is part of the retromer complex and plays an important role in endocytic sorting pathways. It has been shown that at least two SNX27 interacting partners, GLUT1 and TANC2, are aberrantly trafficked due to the E6 PBM-dependent interaction with SNX27. To investigate further which other components of the endocytic trafficking pathway might be affected by the SNX27-HPV E6 interaction, we analyzed the SNX27 proteome interaction profile in a previously described HeLa cell line expressing GFP-SNX27, both in the presence and absence of the HPV-18 E6 oncoprotein. In this study, we identify a novel interacting partner of SNX27, secreted glycoprotein EMILIN2, whose release is blocked by HPV18 E6 in a PBM-dependent manner. Mechanistically, E6 can block EMILIN2 interaction with the WNT1 ligand, thereby enhancing WNT1 signaling and promoting cell proliferation. IMPORTANCE: This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

Exploring the Role of E6 and E7 Oncoproteins in Cervical Oncogenesis through MBD2/3-NuRD Complex Chromatin Remodeling.

BACKGROUND: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. METHODS: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. RESULTS: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. CONCLUSIONS: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

In vitro study of HPV18-positive cervical cancer HeLa cells based on CRISPR/Cas13a system.

The E6 protein is a known oncogene in cervical cancer and plays a key role in the development and progression of cervical cancer by reducing the expression level of the tumor suppressor protein P53 and ultimately leading to enhanced cell proliferation and reduced apoptosis. Therefore, antiviral agents that inhibit the expression of E6 oncoprotein are expected to be potential therapies for human cervical cancer. Here we developed CRISPR/Cas13a: crRNA dual plasmid system and demonstrated that CRISPR/Cas13a could effectively and specifically knock down human papillomavirus 18 E6 mRNA, downregulate the expression level of E6 protein, and restore the expression of the tumor suppressor gene P53 protein, thereby inhibiting the growth of cervical cancer cells and increasing their apoptosis, the E6-2, E6-3, and E6-5 groups resulted in apoptosis rates of 25.4%, 22.4%, and 22.2% in HeLa cells. Moreover, CRISPR/Cas13a enhances the proliferation inhibition and apoptosis induction of cisplatin in cervical cancer HeLa cells. The CRISPR/Cas13a system targeting HPV E6 mRNA may be a promising therapeutic approach for the treatment of human papillomavirus-associated cervical cancer.

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels and activates the DNA damage response.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK + HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting-dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting-dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild-type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. E2-TopBP1 interaction promotes mitotic acetylation of CHK2, promoting phosphorylation and activation of the DNA damage response (DDR). The results present a new model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis, and activates the DDR. This is a novel mechanism of HPV16 activation of the DDR, a requirement for the viral life cycle. IMPORTANCE: Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

Immunological Response against Breast Lineage Cells Transfected with Human Papillomavirus (HPV).

Breast cancer is the most common neoplasm worldwide. Viral infections are involved with carcinogenesis, especially those caused by oncogenic Human Papillomavirus (HPV) genotypes. Despite the detection of HPV in breast carcinomas, the virus's activity against this type of cancer remains controversial. HPV infection promotes remodeling of the host's immune response, resulting in an immunosuppressive profile. This study assessed the individual role of HPV oncogenes in the cell line MDA-MB-231 transfected with the E5, E6, and E7 oncogenes and co-cultured with peripheral blood mononuclear cells. Immunophenotyping was conducted to evaluate immune system modulation. There was an increase in CD4+ T cell numbers when compared with non-transfected and transfected MDA-MB-231, especially in the Treg profile. Pro-inflammatory intracellular cytokines, such as IFN-γ, TNF-α, and IL-17, were impaired by transfected cells, and a decrease in the cytolytic activity of the CD8+ and CD56+ lymphocytes was observed in the presence of HPV oncogenes, mainly with E6 and E7. The E6 and E7 oncogenes decrease monocyte expression, activating the expected M1 profile. In the monocytes found, a pro-inflammatory role was observed according to the cytokines released in the supernatant. In conclusion, the MDA-MB-231 cell lineage transfected with HPV oncogenes can downregulate the number and function of lymphocytes and monocytes.

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

Field experience with the 8-HPV-type oncoprotein test for cervical cancer screening among HPV-positive women living with and without HIV in LMICs.

Overexpression of HPV-oncoproteins E6 and E7 is necessary for HPV-driven cervical carcinogenesis. Hence, these oncoproteins are promising disease-specific biomarkers. We assessed the technical and operational characteristics of the 8-HPV-type OncoE6/E7 Cervical Test in different laboratories using cervical samples from HPV-positive women living with (WLWH) and without HIV. The 8-HPV-type OncoE6/E7 Test (for short: "OncoE6/E7 test") was performed in 2833 HIV-negative women and 241 WLWH attending multicentric studies in Latin America (ESTAMPA study), and in Africa (CESTA study). Oncoprotein positivity were evaluated at each testing site, according to HIV status as well as type-specific agreement with HPV-DNA results. A feedback questionnaire was given to the operators performing the oncoprotein test to evaluate their impression and acceptability regarding the test. The OncoE6/E7 test revealed a high positivity rate heterogeneity across all testing sites (I2: 95.8%, p < .01) with significant lower positivity in WLWH compared to HIV-negative women (12% vs 25%, p < .01). A similar HPV-type distribution was found between HPV DNA genotyping and oncoprotein testing except for HPV31 and 33 (moderate agreement, k = 0.57). Twenty-one laboratory technicians were trained on oncoprotein testing. Despite operators' concerns about the time-consuming procedure and perceived need for moderate laboratory experience, they reported the OncoE6/E7 test as easy to perform and user-friendly for deployment in resource-limited settings. The high positivity rate variability found across studies and subjectivity in test outcome interpretation could potentially results in oncoprotein false positive/negative, and thus the need for further refinements before implementation of the oncoprotein testing in screen-triage-and-treat approaches is warranted.

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.

Structural insights into the functional mechanism of the ubiquitin ligase E6AP.

E6AP dysfunction is associated with Angelman syndrome and Autism spectrum disorder. Additionally, the host E6AP is hijacked by the high-risk HPV E6 to aberrantly ubiquitinate the tumor suppressor p53, which is linked with development of multiple types of cancer, including most cervical cancers. Here we show that E6AP and the E6AP/E6 complex exist, respectively, as a monomer and a dimer of the E6AP/E6 protomer. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6. The extended α1-helices of the dimer intersect symmetrically and contribute to the dimerization. The two protomers sway around the crossed region of the two α1-helices to promote the attachment and detachment of substrates to the catalytic C-lobe of E6AP, thus facilitating ubiquitin transfer. These findings, complemented by mutagenesis analysis, suggest that the α1-helix, through conformational transformations, controls the transition between the inactive monomer and the active dimer of E6AP.

Human papillomavirus-16 E6 activates the pentose phosphate pathway to promote cervical cancer cell proliferation by inhibiting G6PD lactylation.

High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. Here, we report that HPV16 E6E7 promotes cervical cancer cell proliferation by activating the pentose phosphate pathway (PPP). We found that HPV16 E6 activates the PPP primarily by increasing glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. Mechanistically, HPV16 E6 promoted G6PD dimer formation by inhibiting its lactylation. Importantly, we suggest that G6PD K45 was lactylated during G6PD-mediated antioxidant stress. In primary human keratinocytes and an HPV-negative cervical cancer C33A cells line ectopically expressing HPV16 E6, the transduction of G6PD K45A (unable to be lactylated) increased GSH and NADPH levels and, correspondingly, decreasing ROS levels. Conversely, the re-expression of G6PD K45T (mimicking constitutive lactylation) in HPV16-positive SiHa cells line inhibited cell proliferation. In vivo, the inhibition of G6PD enzyme activity with 6-aminonicotinamide (6-An) or the re-expression of G6PD K45T inhibited tumor proliferation. In conclusion, we have revealed a novel mechanism of HPV oncoprotein-mediated malignant transformation. These findings might provide effective strategies for treating cervical and HPV-associated cancers.