RESUMEN
Persistent infections with high-risk human papillomaviruses (HR-HPV) from the genus alpha are established risk factors for the development of anogenital and oropharyngeal cancers. In contrast, HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer (cSCC) in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Keratinocytes are the in vivo target cells for HPV, but keratinocyte models to investigate the replication and oncogenic activities of beta-HPV genomes have not been established. A recent study revealed, that beta-HPV49 immortalizes normal human keratinocytes (NHK) only, when the viral E8^E2 repressor (E8-) is inactivated (T. M. Rehm, E. Straub, T. Iftner, and F. Stubenrauch, Proc Natl Acad Sci U S A 119:e2118930119, 2022, https://doi.org/10.1073/pnas.2118930119). We now demonstrate that beta-HPV8 and HPV38 wild-type or E8- genomes are unable to immortalize NHK. Nevertheless, HPV8 and HPV38 express E6 and E7 oncogenes and other transcripts in transfected NHK. Inactivation of the conserved E1 and E2 replication genes reduces viral transcription, whereas E8- genomes display enhanced viral transcription, suggesting that beta-HPV genomes replicate in NHK. Furthermore, growth of HPV8- or HPV38-transfected NHK in organotypic cultures, which are routinely used to analyze the productive replication cycle of HR-HPV, induces transcripts encoding the L1 capsid gene, suggesting that the productive cycle is initiated. In addition, transcription patterns in HPV8 organotypic cultures and in an HPV8-positive lesion from an EV patient show similarities. Taken together, these data indicate that NHK are a suitable system to analyze beta-HPV8 and HPV38 replication. IMPORTANCE High-risk HPV, from the genus alpha, can cause anogenital or oropharyngeal malignancies. The oncogenic properties of high-risk HPV are important for their differentiation-dependent replication in human keratinocytes, the natural target cell for HPV. HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Currently, the replication cycle of beta-HPV has not been studied in human keratinocytes. We now provide evidence that beta-HPV8 and 38 are transcriptionally active in human keratinocytes. Inactivation of the viral E8^E2 repressor protein greatly increases genome replication and transcription of the E6 and E7 oncogenes, but surprisingly, this does not result in immortalization of keratinocytes. Differentiation of HPV8- or HPV38-transfected keratinocytes in organotypic cultures induces transcripts encoding the L1 capsid gene, suggesting that productive replication is initiated. This indicates that human keratinocytes are suited as a model to investigate beta-HPV replication.
Asunto(s)
Virus del Papiloma Humano , Queratinocitos , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Epidermodisplasia Verruciforme/virología , Queratinocitos/virología , Neoplasias de Células Escamosas/virología , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Virus del Papiloma Humano/genética , Genoma ViralRESUMEN
Beta-2 Human papillomaviruses 38b, 107, and 122 have been frequently found in cervical cancer samples in western Mexico. Because their E6/E7 genes functions are not fully elucidated, we deepen into their transformation capabilities. To achieve this goal, primary human fibroblasts (FB) were transduced with E6/E7 genotype-specific viral particles. Additionally, E6/E7 from HPVs 16 and 18 were included as controls. All E6/E7-cell models increased their lifespan; however, it is important to highlight that FB-E6/E7-122 showed growth as accelerated as FB-E6/E7-16 and 18. Furthermore, both FB-E6/E7-38b and 122 exhibited abilities to migrate, and FB-E6/E7-122 presented high invasive capacity. On the other hand, ΔNp73 expression was found in all cell models, except for FB-pLVX (empty-vector). Finally, RNAseq found differentially expressed genes enriched in signaling pathways related to cell cycle, epithelial-mesenchymal transition, and cancer, among others. This study shows for the first time, the great transformative potential that genotypes of the Beta-2 also possess, especially HPV122. These Beta-2 HPVs can modulate some of the genes that are well known to be regulated by Alpha-HPVs, however, they also possess alternative strategies to modulate diverse signaling pathways. These data support the idea that Beta-2 HPVs should play an important role in co-infections with Alpha-HPV during carcinogenesis.
Asunto(s)
Proteínas Oncogénicas Virales , Neoplasias del Cuello Uterino , Femenino , Fibroblastos/metabolismo , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Represoras/metabolismo , Neoplasias del Cuello Uterino/genéticaRESUMEN
Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.
Asunto(s)
Transformación Celular Neoplásica/patología , Activación Enzimática/efectos de la radiación , Queratinocitos/metabolismo , Papillomaviridae/crecimiento & desarrollo , Proteínas E7 de Papillomavirus/metabolismo , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/efectos de la radiación , Proteínas Virales/metabolismo , Proliferación Celular/genética , Células Cultivadas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Piel/parasitología , Piel/virología , Neoplasias Cutáneas/virología , Receptor Toll-Like 9/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
IMPORTANCE: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.