Determination of the in vitro antiviral activity of an engineered M1GS ribozyme that targets to the core gene of hepatitis C virus

Abstract:

Abstract:［Objective］Hepatitis C virus (HCV) is one of the major pathogens that lead to viral hepatitis． At present，Interferon treatment in combination with ribavirin is the first line clinical therapeutic approach． However，the responses are usually poor and the viral infection reoccurs． Therefore，exploring new antiviral agents and therapies is under urgent needs．［Methods］The sequence and structure of the core coding region of HCV genome were analyzed through the two computer software，DNAMAN and RNA Structure． The cytosine 52 nt downstream of the AUG initiation triplet was identified as the optimal target cleavage site． Based on the flanking sequence of this assumed cleavage site，a guide sequence (GS) was designed and covalently linked to the 3 prime terminus of the M1 RNA，which is catalytic subunit of the RNase P derived from Escherichia coli using PCR． We named this new targeting ribozyme M1GS-HCV /C52 and it antiviral activities were analyzed in cultured cells．［Results］In the in vitro cleavage assay，M1GS-HCV /C52 ribozyme could effectively cleave the HCV target RNA into two fragments at the specific cleavage site． Moreover，comparing to the blank control，this engineered M1GS ribozyme could reduce the core protein expression of more than 80% in the HCVinfected host cell and lead to a 1500-fold reduction of HCV RNA copies in the culture supernatant． An another M1GS ribozyme，M1GS-HCV /C52 * ，which has the same guide sequence but does not contain a 24nt-long bridge sequence，did not exhibit apparent inhibition for the expression of HCV core gene and viral proliferation in our paralleled assay．［Conclusion］We successfully constructed an M1GS ribozyme showing affective and specific cleavage of target viral RNA．Further results showed that the engineering ribozyme had notably antiviral activity in cultured cells，thus provided a new promising approach for clinical anti-HCV therapeutic strategy.