Whole genome analysis of cells permissive for late gene expression of HPV-16

Aim was to identify cellular factors that regulate HPV-16 late gene expression at the level of RNA processing Cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5’-splice site SD3632, produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c) and HuR that are known to regulate HPV16 late gene expression, or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the hnRNP C-family. Overexpression of RALYL, or RALY and hnRNP C1 that are two other members of the hnRNP C-family, induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. Induction of HPV16 late gene expression by the hnRNP C-proteins was dependent on the HPV16 early untranslated region to which these proteins also were shown to bind in vitro, and in living cells. Our experiments revealed that hnRNP C proteins that interacted with the HPV16 early untranslated region reached out to the splicing silencer complex at HPV16 SD3632 and derepressed this splice site, thereby activating production of HPV16 spliced late L1 mRNAs. In conclusion, hnRNP C- proteins bind to the HPV16 early untranslated region and control of HPV16 late L1 mRNA splicing. Total cellular RNA was extracted from stable cell lines. Samples were prepared in triplicates. C33ARSVNeo served as control cell line.