The Human Papillomavirus Type 16 E5 Protein: Stress Signaling and Expression in Cervical Cancer Cell Lines
Sudarshan, Sawali Ranganath
Riegel, Anna T
ABSTRACTHuman papillomavirus (HPV) is the causative agent of 99% of cervical cancers, approximately 60% of which are caused by the high-risk type 16. While two of the three HPV-16 oncoproteins, E6 and E7, have been well characterized, the biological functions of the E5 protein remain elusive. HPV-16 E5 protein (16E5) is thought to be disrupted during viral integration, a key event in cellular transformation, suggesting that 16E5 is not essential for carcinogenesis. Conversely, its presence has been shown to enhance E6 and E7-induced transformation, and several studies, including those by Sherman et. al. in 1992 and Bauer-Hofmann et. al. in 1996, have shown the presence of potentially E5-coding transcripts even in cervical cancer cell lines with integrated viral genomes. However, direct confirmation of 16E5 expression has not been possible, mostly attributable to the lack of specific antibodies. Since there is no 16E5 antibody available, confirming protein expression in cancer cells has not been possible. In this thesis, we use mass spectrometry to identify 16E5 in two HPV-16 positive cervical cancer cell lines, SiHa and CaSki. This is the first time that 16E5 has been identified in cervical cancer lines with an integrated HPV genome, and our results demonstrate the co-expression of E5 later in malignancy when E6 and E7 are highly expressed. In addition, we analyze gene expression changes induced by the endoplasmic reticulum (ER)-resident E5 in human foreskin keratinocytes (HFKs) using microarray and real-time RTPCR. We identify the downregulation of spliced XBP-1, IRE1;, and COX-2, key players in the ER-stress response, as biochemical markers of E5 expression. Similar studies were performed in HFKs expressing either low-risk HPV-6b E5 or a C-terminal 16E5 mutant, where the histidine and alanine residues at position 77 and 78 (conserved in high-risk HPVs) were replaced with tyrosine and isoleucine (conserved in low-risk HPVs). Importantly, neither construct affected levels of these genes, suggesting that the ability to induce these changes in stress gene expression is specific to 16E5 and might facilitate viral persistence leading to cell transformation. Overall, the data presented within this thesis suggest a potential role for E5 in productive viral infection as well as the initial and late stages of malignant transformation.
Showing items related by title, author, creator and subject.
Castellsague, Xavier; Bosch, Xavier; Munoz, Nubia; Meijer, Chris J. L. M.; Shah, Keerti V.; de Sanjose, Silvia; Eluf-Neto, Jose; Ngelangel, Corazon A.; Chichareon, Saibua; Smith, Jennifer S.; Herrero, Rolando; Franceschi, Silvia (International Agency for Research on Cancer Multicenter Cervical Cancer Study Group, 2002-04-11)