Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand

Department of Pathology and Laboratory Medicine, The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia-St. Paul's Hospital, Vancouver, British Columbia, Canada.
Journal of Virology (Impact Factor: 4.65). 03/2005; 79(4):2151-9. DOI: 10.1128/JVI.79.4.2151-2159.2005
Source: PubMed

ABSTRACT Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

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    • "RNAi is an effective tool to inhibit specific gene expression , espe - cially in the pathogenesis of viral infections ( Yao et al . , 2012 ; Yuan et al . , 2005 ) . RNAi inhibition depends on the efficacy of delivery of siRNA or shRNA . In our study , we inhibited the ITK expres - sion by using an ITK - specific siRNA , in vitro . The pGCSIL plasmid used in our in vivo research contains a U6 promoter , and contin - uously expressed shRNA . The pGCL - shITK functioned well in vivo in our previou"
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    • "The successful examples for inhibition of viral pathogens of myocarditis includes deliveries of i) ASONs targeting CVB3 IRES (Yuan et al., 2006) and both ends of the CVB3 genome (A. Wang et al., 2001), ii) siRNAs targeting CVB3 2A (Merl et al., 2005; Yuan et al., 2005) and 3D (Ahn et al., 2005; Schubert et al., 2005; Schubert et al., 2007), iii) plasmids expressing shRNAs targeting 3D and VP1 (J. Y. Kim et al., 2007) and vi) ribozymes targeting HCV RNA (Gonzalez-Carmona et al., 2006). "
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    • "The vast majority of siRNAs developed against EV focus upon the degradation of mRNAs coding for the viral RNA-dependent RNA polymerase (RdRp) and proteases. For example, siRNAs directed against protease 2A were the most effective in the inhibition of CVB3 infection in HeLa cells and murine cardiomyocytes (Yuan et al., 2005). Interferon receptor knock-out mice transfected with siRNAs targeting CVB3 protease 2A exhibited increased survival time and attenuated viral replication when challenged with CVB3 (Merl et al., 2005). "
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