engleski

Inhibition of HCV RNA Synthesis By RNA Structural Mimicry in Spite of Substantial Sequence Differences

Potent sequence-specific inhibition of HCV RNA is possible using RNA interference (RNAi) technology. However, the problem of viral RNA inaccessibility, and the escape of genetic mutants due to high mutation rates of HCV remains. AIM: The objective of this study was to develop an alternative RNA-based approach by over expression of RNAs that mimic secondary structure, but without identical sequences corresponding to cis-acting replication elements of HCV RNA 3'-terminal regions. METHODS: Structural mimics were constructed based on RNA sequences of the HCV 1b subgenomic replicon, BB7. The HCV mimic RNA sequences were predicted to adopt stem-loop structures identical to the corresponding cis-acting replication element in full-length viral RNA, using mfold ver 3. DNA fragments containing mimic sequences flanked by BamH I and Hind III sites were generated by amplification of sequences from pHCV replbBB7 and verified by sequencing. To determine effects of structural mimics on HCV infection, a Huh 7.5 cell line was infected with HCV-JFH 1 virus. Cells supporting stable propagation of JFH virus after 5 days of infection were transfected with expression vectors generating HCV structural mimics. Levels of cellular HCV RNA converted to DNA were quantitated 2 days posttransfection by Real-Time SYBR Green PCR using HCV specific primers, and normalized to LDH A mRNA levels. An HBV-specific structural mimic was a negative control. Cellular HCV protein levels were determined by Western blots using anti-NS3 specific antibody (Biodesign, Saco, ME)and anti-tubulin antibody (Abcam Inc, Cambridge, MA).To evaluate possible antisense effects, the 5B RNA mimic was divided into small antisense RNA molecules, and transfected into HCV-JFH 1 infected Huh 7.5 cells. RESULTS: Compared to negative control, HBV mimic, the 5B mimic decreased expression of HCV by 82% by Real-Time PCR. The decreases in HCV mRNA were confirmed by decreases in NS3 protein compared to negative control and standardized by the expression of housekeeping protein, tubulin by Western blot. Small RNA molecules, as a control for possible antisense effect, decreased expression of HCV by 4%, not significantly different to negative control. CONCLUSION: These results suggest that RNAs that mimic HCV structures corresponding to cis-acting replication elements of HCV RNA in spite of substantial sequence differences can inhibit HCV RNA replication. This strategy may circumvent the problem of viral RNA inaccessibility and escape of genetic variants.

RNA interference ; 3'-terminal regions ; HCV infection ; JFH virus ; cis-acting replication elements

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