Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 03 Aug 2019 15:04:33 Determination of human papillomavirus 16 physical status through E1/E6 and E2/E6 ratio analysis Dimitris Tsakogiannis, 1 Zaharoula Kyriakopoulou, 1 Irina Georgia Anna Ruether, 1 Grigoris D. Amoutzias, 1 Tilemachos G. Dimitriou, 1 Valentina Diamantidou, 1 Constantin Kotsovassilis 2 and Panayotis Markoulatos 1 Correspondence Panayotis Markoulatos markoulatos@bio.uth.gr Received 7 April 2014 Accepted 10 September 2014 1 Microbiology–Virology Laboratory, Department of Biochemistry and Biotechnology, School of Health Sciences, University of Thessaly, Larissa, Greece 2 Clinical Biochemistry Department, General Hospital of Athens, Athens, Greece Human papillomavirus (HPV) 16 genome integration into the host chromosome is a crucial event during the life cycle of the virus and a major step towards carcinogenesis. The integration of HPV16 DNA promotes a constitutive high expression level of E6 and E7 oncoproteins, resulting in the extensive proliferation of the infected epithelial cells. In the present report the physical status of the HPV16 genome was studied, through determination of E1/E6 and E2/E6 DNA copy number ratios in 61 cervical samples of low- and high-grade malignancy and 8 cervical cancer samples, all of them associated with HPV16 infection. The selection of E1, E2 and E6 amplification target regions was performed according to the most prevalent deleted/disrupted sites of E1 and E2 genes. For this target selection we also considered the most conserved regions of E1, E2 and E6 genes among the same HPV16 isolates that were recently reported by our group. The analysis of HPV16 DNA form revealed a significant association among the mixed DNA forms in low-grade and high-grade malignancies, (x 2 , P,0.01). The comparative analysis of E1/E6 and E2/E6 in the same cervical samples provides an accurate picture of HPV16 DNA form and may reveal whether different HPV16 DNA integrants coexist in the same cervical sample or not. This study proposes that E1/E6 and E2/E6 ratios determine with accuracy the HPV16 DNA integration pattern and may predict multiple integration events in the examined sample, thus providing significant information about the progression of cervical dysplasia. INTRODUCTION Cervical cancer is the third most common type of cancer among women worldwide, with a high mortality rate. The worldwide incidence of cervical carcinoma is more than 530 000 cases per year, whereas mortality reaches 275 000 deaths annually, of which approximately 85 % occur in developing countries (Jemal et al., 2011; Forman et al., 2012). The aetiological agents for the development of high- grade precancerous cervical lesions and invasive cervical cancer are the oncogenic human papillomavirus (HPV) types present as persistent infections (Mun ˜oz et al., 2003). To date, more than 150 different HPV types have been characterized and about 40 of them are related with anogenital tract malignancy, grouped as high-risk (HR) or low-risk (LR) genotypes (zur Hausen, 1996; Bernard et al., 2006, 2010). Epidemiological studies revealed that HPV16 is the most commonly observed HR HPV type, followed by HPV18, 31, 33 and 45 (de Sanjose et al., 2010; Li et al., 2011). Persistent infection with HR HPV types is associated with an increasing risk of integration of the viral circular genome (episome) into the host chromosomes, leading to cancer development. The circular HPV genome is then linearized, but the long control region, and the E6 and E7 oncogenes, are always retained intact (Wentzensen et al., 2004; Xu et al., 2013; Akagi et al., 2014). Viral integration appears to coincide with the development of high-grade cervical intraepithelial neoplasia (CIN II, III) as a consequence of overexpression of the E6 and E7 oncogenes (Doorbar et al., 2012). In particular, the integra- tion of the HPV16 genome usually disrupts the E2 and/or E1 regions found downstream of the early genes E6 and E7, thus resulting in functional inactivation of the E1 and E2 proteins (Chen et al., 1994; Arias-Pulido et al., 2006; Cricca et al., 2009). Disruption events within the E1 and/or E2 genes lead to interference of viral DNA replication control. Moreover, disruption of the E2 gene results in the Abbreviations: CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; HR, high-risk; RT-PCR, real-time PCR. Journal of Medical Microbiology (2014), 63, 1716–1723 DOI 10.1099/jmm.0.076810-0 1716 076810 G 2014 The Authors Printed in Great Britain