engleski

Inhibition of HCV RNA synthesis by the expression of RNA structural mimicry

HCV infection is a difficult health problem worldwide. A number of strategies are being studied for their potential in future therapy. Potent sequence-specific inhibition of RNA is now available in the form of RNA interference (RNAi) technology. However, the problem of viral RNA inaccessibility, and the escape of genetic mutants due to high mutation rates of HCV virus still remains. AIM: The objective of this study was to develop an alternative RNA-based approach by over expression of mimicry RNAs with a secondary structure corresponding to cis-acting replication elements of HCV RNA 3-terminal regions. The lack of dependence on sequence complementarity could result in inhibition of viral RNA replication while minimizing the development mutation driven resistance. Methods: Structural mimics were constructed based on RNA sequences of the HCV 1b subgenomic replicon, BB7. The HCV RNA sequence was predict 5B RNA mimic to adopt stem-loop structures identical to the corresponding cis-acting replication element in full-length viral RNA (7600- 7694 nt, NS5B coding region SL 3.1, 3.2) using mfold ver 3.1. 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, an 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 from pSilencer 4.1 CMV for 48 hrs. Cellular HCV RNA levels were quantitated by Real-Time SYBR Green PCR using HCV specific primers. Lactate dehydrogenase A mRNA levels in each sample was used to normalize JFHv cDNA levels. A HBV specific structural mimic was used as a negative control . Western blot analysis was performed using anti-HCV antibodies to determine if the 5B RNA mimic could reduce levels of HCV proteins. Results: An 8-fold reduction of HCV RNA in Huh 7.5 cells infected with HCV-JFHv-1 was seen on day 2 after HCV 5B RNA mimic treatment compared to negative control HBV RNA-mimic treatment by Real-Time PCR. Conclusions: These results suggest that RNAs that mimic HCV structures corresponding to cis-acting replication elements of HCV RNA inhibit viral RNA replication and deserve further evaluation for the treatment of hepatitis C. This strategy may be advantageous over other sequence-specific gene therapy modalities in circumventing the problem of viral RNA inaccessibility and the escape of genetic variants.

HCV infection ; RNA interference (RNAi) ; Huh 7.5 cell line ; HCV 1b subgenomic replicon BB7

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