Biological and biochemical properties of natural variants of HPV-18

HPV-16 and HPV-18 infections are strongly associated with the risk of developing cervical cancer. Worldwide, HPV-16 type is the most prevalent type in invasive squamous cell carcinomas of the cervix followed by HPV-18, whereas both viral types are similarly prevalent in adenocarcinoma cases. HPV-18 intratypic nucleotide variability has been studied resulting in important findings concerning the evolution and phylogeny of the virus and the natural history of infections. Although the studies about the clinical relevance of HPV-18 genetic variability are very limited, overall, it is suggested that Amerindian (As+AI) and European (E) variants represent isolates with a higher oncogenic potential compared to the African (Af) variants. Furthermore, it was observed that HPV-18 Af variants are exclusively detected in samples of invasive squamous cell carcinoma of the cervix, whereas As+AI and E variants are more prevalent in adenocarcinoma and adenosquamous cell carcinoma. With regard to biological differences among HPV-18 variants, it is crucial to emphasize that the few studies conducted so far explored only the variability of the viral E6 oncoprotein, and most importantly, none of the studies was performed in HPV-18 natural host cell models, which are primary human keratinocytes (PHK). For these reasons, we characterized functionally PHKs immortalized by two different variants of HPV-18. PHKs were transduced with E6/E7 of HPV-18 As+AI and Af variants (PHK18AA and PHK18Af, respectively), and subcultured until p30, when these were considered immortalized. PHK18AA reached immortalization significantly faster than PHK18Af, even though a \"crisis\" was not observed in any of the transduced PHKs. Nevertheless, there were not proliferation rates difference among PHK18AA e PHK18Af at p30. However, immortalized PHK18AA were more efficient in forming colonies and spheroids in monolayer and three-dimensional cultures, respectively, using different assays. In addition, immortalized PHK18AA showed greater efficiency in invading through a collagen matrix, although no differences in migration were observed among variants. We also observed that baby rat kidney cells (BRK) co-transfected with E7 Af and RAS generated a smaller number of colonies, which may be partly associated with the inability of E7 Af to form a complex with the ubiquitin ligase UBR4, and further degrade the tumor suppressor protein PTPN14. On the other hand, immortalized PHK18Af showed greater resistance to differentiation in a medium enriched with serum and calcium. Finally, raft cultures derived from PHKs immortalized by both HPV-18 variants were morphologically similar and showed equivalent levels of proteins associated with cell differentiation and proliferation. Taken together, our data indicates that the HPV-18 As+AI variant has a greater oncogenic potential compared to the Af counterpart, in addition to interacting differently with some cellular targets. This study is unique in analyzing the biological and biochemical of E6/E7 oncoproteins of natural variants of HPV-18 in the context of the natural host, that is, epithelial cells (AU)