Trans-targeting of human papillomavirus type-16 E6 and E7 transcripts by an engineered hairpin ribozyme
Siwkowski, Andrew M.
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Three hairpin ribozymes have been designed which cleave sequences from HPV-16 E6 and E7 transcripts in vitro. E6 and E7 proteins from high risk HPV types are capable of immortalizing human cells and are therefore considered transforming proteins. Two of these ribozymes (RHPV419 and RHPV434) are derivatives of the hairpin ribozyme which occurs in the minus strand of the tobacco ringspot virus satellite RNA [(-)sTRSV]. The third ribozyme (RHPV195) was derived from the hairpin ribozyme which occurs in the minus strand of the chicory yellow mottle virus satellite RNA 1 [(-)sCYMVl], This marks the first time that the (-)sCYMVl derived hairpin ribozyme has been shown capable of being adapted for trans cleavage of nonnative substrate RNA. The helix-1 lengths of these three ribozymes have been optimized for maximum cleavage efficiency. Michaelis-Menten kinetic parameters have been determined for both (-)sTRSV hairpin ribozyme derivatives. RHPV434 was tested using three different helix-4/loop-3 variants, and the variant supporting the highest cleavage activity was used for further development. A derivative of RHPV434, tVl-434 which contains a 5' tRNAva^ and a 3' hairpin loop structure, was found fully active in vitro compared to the unmodified RHPV434. A mismatch was introduced into helix-1 of RHPV419 in order to disrupt a potential RNA pol III termination sequence and the helix-1 length was reoptimized. RHPV419 and RHPV434 were cloned into plasmids containing RNA pol III promoter sequences. RHPV434 was also cloned into a retroviral vector for studies on ribozyme activity in a human cell line transformed by HPV-16. To determine ribozyme efficacy in these and future experiments, a tripartite assay system was developed which consists of evaluating cell morphology, target level, and ribozyme expression.