Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response

Abstract

Integration of high-risk human papillomavirus (HRHPV) into the host genome is a key event in cervical neoplastic progression. Integration is associated with deregulated expression of the viral oncogenes E6 and E7 and acquisition of a selective growth advantage for cells containing integrants. Overexpression of the viral transcriptional regulator E2 from heterologous promoters has an inhibitory effect on transcription from integrated HRHPV. Therefore, we hypothesized that loss of E2-expressing episomes from cells in which integration had previously occurred would be required for such cells to gain a growth advantage. Using the unique W12 model of cervical squamous carcinogenesis, we show that cells containing integrated HPV16 reproducibly emerged during long-term culture when there had been a rapid fall in episome numbers. During the period of emergence, it is possible to isolate single-cell clones containing an intracellular mixture of the integrant being selected and episomes at reduced load. The lower level of E2 expression seen in such cells is associated with partial inhibition of transcription from the HPV16 integrant. Full deregulation is not observed until complete loss of E2-expressing episomes occurs. Microarray analysis showed that episome loss was closely associated with endogenous activation of antiviral response genes that are also inducible by the type I IFN pathway. Taken together, our results indicate that episome loss, associated with induction of antiviral response genes, is a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. We conclude that cervical carcinogenesis requires not only HRHPV integration, but also loss of inhibitory episomes.human papillomavirus ͉ cervix ͉ integration ͉ interferon ͉ progression I ntegration of high-risk human papillomavirus (HRHPV) into the host genome is an important step in cervical neoplastic progression (1, 2). Integrated viral genomes from which HRHPV early genes are transcribed have been detected in Ϸ87.5% of cervical malignancies (3). Integration usually causes deletion or disruption of the viral regulatory E2 gene, while retaining a variable segment including the E6 and E7 oncogenes and the upstream regulatory region (4, 5). Overexpression of E2 from heterologous promoters in cells harboring integrated HRHPV can repress the early promoter of the integrated virus, causing a sharp reduction in E6 and E7 expression (6). Thus, HRHPV integration and disruption͞deletion of E2 leads to increased expression of the viral oncogenes (7,8). Cells containing integrated HRHPV acquire a growth advantage over cells harboring episomal HRHPV (the natural viral state in productive infections) and show increased genomic instability (9-11).Cervical keratinocyte cell lines established from precursor low-grade squamous intraepithelial lesions have indicated that episomal HRHPV genomes are maintained at Ϸ100 copies per cell in the basal region of an infected epithelium (12, 13). Viral integration is therefore most likely to occur in cel...

Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma

“…43 We demonstrated the integrated form of HPV 16 in all of the carcinoma specimens and significantly increased HPV 16 integration in cervical carcinoma compared with CIN 2/3, supporting the notion that HPV 16 integration into the host genome is an important step of carcinogenesis. 17,18 In most published studies of cervical carcinoma, the integrated forms of HPV 16 were also observed in either all or a majority of the specimens. 42,43 AriasPulido et al 22 recently conducted a study of formalin-fixed paraffin-embedded as well as fresh cervical specimens using qRT-PCR and reported a relatively low proportion of the integrated type of HPV 16 in carcinoma in situ (30.3%) and in invasive carcinoma (60.9%).…”

“…In the second step of our screening process, specimens positive for b-globin were screened for HPV by using GP5 þ /GP6 þ consensus primermediated PCR, which can detect 14 oncogenic HPV genotypes, including the eight major oncogenic HPV types (16,18,31,33,35,45,52, and 58). PCR was performed according to Jacobs et al 32 A 25-ng aliquot of genomic DNA was added to the PCR master mixture, which contained 1 Â PCR buffer, 200 mM each deoxynucleoside triphosphate, 200 nM primer, 3.5 mM MgCl 2 , and 2.5 U of AmpliTaq Gold DNA polymerase.…”

“…4,8 Munoz and Clifford, in separate meta-analyses of pooled data, concluded that eight genotypes of oncogenic HPV (16,18,31,33,35,45,52, and 58) are closely associated with more than 80% of cases of cervical carcinoma and CIN 2/3. HPV 16 alone was detected in 54-55% of cervical carcinomas and 45% of CIN 2/3.…”

“…[11][12][13][14][15][16] Integrating HPV 16 into the host genome of infected cells is considered a critical step for carcinogenesis. 17,18 HPV 16 integration promotes the transcription of E6 and E7 viral oncogenes. This occurs by disruption or deletion of the viral E1 and/or E2 open reading frame, which releases the suppressive effect of the E2 protein on the viral oncogenes E6 and E7, leading to the activation of E6 and E7 transcription.…”

Human Papillomavirus: Biology and Pathogenesis

“…Infected cells with episomal HPV are cleared after exposure to IFN-β, but cells with integrated HPV-DNA are resistant to this antiviral effect (Pett et al, 2006). T-cell response to E2 and E6 are lost or reduced in CIN 3 and carcinoma invasive.…”