Posttranscriptional regulation of transformation by human papillomavirus type 16 E7 and expression of this oncogene

Belaguli, Narasimhaswamy S. (1993) Posttranscriptional regulation of transformation by human papillomavirus type 16 E7 and expression of this oncogene. Doctoral (PhD) thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (36MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

Many epidemiological and experimental studies have strongly implicated human papillomavirus type 16 (IIPV-16) in cervical neoplasia. The oncogenic potential of this virus can be demonstrated by transformation of primary baby rat kidney (BRK) cells by cotransfection of the viral genome with the activated EJ-ras oncogene. I performed a mutational analysis of the viral genome to map the regions essential for its transforming activity. For the SV40-bascd early region expression plasmids, the disruption of the E6, E2, E5 and the 3' region of the El open reading frames (ORFs) did not affect the transforming activity of mutated plasmids, whereas the insertion of translation termination linkers within the E7 ORE abolished transformation. Additional sequences present in the 3'-flanking region of the E7 ORF were also required for efficient transformation. -- The 3' flanking region sequences were analyzed in detail in SV40-based E7 expression plasmids by progressive deletion analysis and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within this 3'-flanking region abolished transformation. Regeneration of the wild- type sequence in a previously transformation incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in higher levels of transformation, whereas mutating the + 2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the transformation-deficient splice site mutants accumulated lower levels of E7 RNA, primarily because of rapid destabilization of E7 RNA. -- The splice sites present within the E6 ORF were examined for their ability to substitute for the loss of nt 880 splice donor site function. The wild-type E6 splice sites, as well as the heterologous splice sites of the SV40 intron, were able to partially substitute for the nt 880 splice site function. These results indicated that the efficient accumulation of HPV-16 E7 RNA and transformation of BRK cells depend on the presence of an intron in the transcription unit. -- Recent studies have indicated the presence of naturally occurring HPV-16 antisense (AS) RNA in cervical carcinomas. I have detected AS E7 and E7/E1 RNA in Cos-1 cells transiently transfected with SV40-based HPV-16 early region expression plasmids. By deletion mutation analysis, the AS promoter was localized to nt 4031-4338 of HPV-16. Primer extension analysis and RNase protection assays indicated that the AS RNA was initiated from multiple sites in an AT-rich region around nt 4100. The AS promoter was active when cloned downstream of the chloramphenicol acetyl transferase (Cat) gene, giving rise to AS Cat RNA. -- AS E7 RNA was detected in both the nucleus and cytoplasm. The AS RNA formed a duplex with the sense (S) E7 RNA. The presence of AS RNA was correlated with reduced splicing of E7 RNA from the nt 880 splice site and the synthesis of E7 protein.

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