Sequence variation analysis of the E2 gene of human papilloma virus type 16 in cervical lesions from women in Greece

Abstract

The E2 gene of human papilloma virus is expressed at the early stage of the viral life cycle, encoding the E2 transcription factor, and regulates the expression of E6 and E7 oncogenes. Disruption of E2 gene due to viral integration inhibits the transcriptional suppression of the HPV oncogenes, inducing cell proliferation. In the present study, a total of 22 HPV16-positive cytological specimens derived from high- and low-grade cervical intraepithelial lesions were investigated in order to identify sequence variations in the HPV16 E2 ORF. The E2 gene was amplified by PCR using external and internal overlapping sets of primers. Amplicons were cloned and sequenced. Disruption sites were detected in cervical samples diagnosed as high-grade cervical intraepithelial lesions. Moreover, sequence variations were identified in the E2 ORF and specific variations were associated with non-European variants such as African type I, African type II and Asian American. A total of three new sequence variations were identified at positions 2791, 2823 (transactivation domain) and 3361 (hinge region). Distinct phylogenetic branches were formed according to E2 analysis that characterized the different HPV16 variants. It was ascertained that non-European variants are circulating in the Greek population.

Association of an intact E2 gene with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome in HPV16 positive head and neck squamous cell carcinoma

“…Three of these were OPSCCs and two were non-OPSCC. Of the three OPSCC samples, all three involved deletions within the hinge region, which has been reported to harbor deletions in cervical cancer [ 50 – 52 ]. Deletions within the hinge region may hinder nuclear localization of E2 and E2-dependent replication.…”

“…Among the two non-OPSCC tumors, one harbored a deletion in the hinge region and the other a deletion in the transactivation domain. However these E2 sequences were identified as being non-functional due to mutations within the E2 gene (Genebank IDs HM162463.1 and HM162476.1) [ 52 ].…”

Sites of disruption within E1 and E2 genes of HPV16 and association with cervical dysplasia

“…The samples were incubated at 37 ο C overnight, and proteinase K was inactivated at 95 ο C for 15 min. The lysate was removed by a brief spin, and the resulting supernatant was transferred into a new tube [Tsakogiannis et al, ].…”

“…Previous studies investigated the physical state of HPV16 genome through mapping of disrupted sites in E1 and E2 ORFs [Kalantari et al, 1998;Lukaszuk et al, 2003;Arias-Pulido et al, 2006;Cricca et al, 2009]. Interestingly, disruption of E2 gene was associated with high grade cervical malignancies [Arias-Pulido et al, 2006;Cricca et al, 2009;Tsakogiannis et al, 2012]. A previous study by Cricca et al [2009] reported that disruption of both E1 and E2 genes occurred in high grade cervical intraepithelial malignancies whereas in low grade cases only E1 gene disruption was observed.…”

Molecular approaches for HPV genotyping and HPV-DNA physical status

“…Such mechanisms include the methtylation status of HPV DNA (Refs 146, 152, 153) or mutations into the viral genome that change the protein functions of the E2 and E6 proteins and subsequently augmented the viral capacity to transform the epithelium. Such mutations have been described at position 3240 (E2, C3410T) (Refs 154, 155) as well as at position 350 (E6, T350G) (Refs 156, 157, 158).…”