Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity.

Abstract

The "high-risk" human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of human cervical carcinomas. In these cancers, the viral DNAs are often integrated into the host genome so that expression of the El and the E2 genes is lost, suggesting that disruption of these regulatory genes plays an important role in carcinogenic pgression. Previous studies defiming the viral genes affecting PV-16 transformation functions have used the "prototype" viral genome, which was cloned from a human cervical carcinoma and later discovered to harbor a mutation in the El gene. In this study, we have corrected this mutation and have evaluated the effect of mutations of either the El or the E2 gene on the efficiency of HPV-16 immortalization of human keratinocytes. Mutation of either the El gene or the E2 gene in the background of a "wild-type" HPV-16 genome markedly increased immortalization capacity. Mutations were also generated in the E2-binding sites located upstream of the P,7 promoter, which directs synthesis of the viral E6 and E7 transforming genes. E2 negatively regulates the Pg. promoter through bindin at adjacent sites. Surprly, the mutation of these sites only partially relieved the negative effect of E2 on viral immortalization, implicating additional mechaniss in theE2 repression of viral immortalization functions. Our results provide genetic evidence that the El and E2 gene products each can repress HPV-16 immortalization and support the hypothesis that a selective growth advantage is provided by integration of the viral-genome in a manner that causes the loss of expression of either El or E2.A subgroup of human papillomaviruses (HPVs) is associated with genital intraepithelial neoplasias and cervical carcinomas. The two viruses found in most of these lesions are HPV types 16 (HPV-16) and 18 (HPV-18). HPV-16 DNA and HPV-18 DNA can immortalize primary human keratinocytes in culture (1-3) and can induce histological abnormalities in organotypic cultures similar to those seen in clinical lesions in vivo (4, 5). Previous analyses have shown that the expression of both the E6 and E7 genes of the "high-risk" HPVs is necessary for the efficient immortalization of primary human keratinocytes (5-7). Furthermore, the E6 and E7 genes are consistently expressed in the HPV-positive cervical cancers and in cell lines derived from such cancers (8-13), supporting the hypothesis that expression of E6 and E7 plays a role in cervical carcinogenesis. Analyses of the state of the viral DNA in HPV-positive lesions have generally correlated the presence of extrachromosomal viral genomes in benign preneoplastic lesions and of integrated viral DNA in cancers (8,(13)(14)(15)(16) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Sequence variation and physical state of human papillomavirus type 16 cervical cancer isolates from Australia and New Caledonia

“…Engineered E2 mutants, particularly of the transactivation region, increase immortalization capacity 28 and transactivation activity. 29 There have, however, been few investigations of the functional significance of natural E2 variants, apart from a study carried out by Veress et al 8 that showed that the transcriptional transactivation potentials of European and AsianAmerican E2 variants were similar.…”

Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts

“…Maximal E7 levels correlated with the acquisition of structural chromosomal abnormalities (Alazawi et al, 2002;Pett et al, 2004). High oncogene levels are attributed to the functional loss of the viral E2 gene product which acts as an intrinsic repressor of E6/E7 expression (Romanczuk and Howley, 1992). Moreover, viral-cellular fusion transcripts are devoid of the instability elements located in the 3 0 -region of the viral mRNA and may thus have a longer half-life .…”

“…Integration of the viral DNA into the host genome is frequently observed in invasive cervical carcinomas and in a subset of CIN3 and is thought to be one of the driving factors for progression . In particular the fact, that integration frequently disrupts the viral regulatory gene E2 from its own promoter is taken as an argument for a constitutive strong expression of the viral oncogenes (Baker et al, 1987;Romanczuk and Howley, 1992). Moreover, experimental data suggest that cis-acting elements within the flanking cellular sequences can positively modulate viral gene expression (von Knebel Doeberitz et al, 1991).…”

Immunotherapy: An Evolving Paradigm in the Treatment of Advanced Cervical Cancer

“…Interruption of E2, which normally functions as a transcriptional regulator of E6 and E7, leads to up-regulation of E6 and E7, degradation of p53 and pRB, respectively, and ultimately malignant transformation. [27][28][29] …”