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: 4.44). 03/2005; 79(4):2151-9. DOI: 10.1128/JVI.79.4.2151-2159.2005
Source: PubMed


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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Available from: David Chau, Aug 10, 2015
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    • "RNAi-based immunity against viral infection is dependent upon Dicer recognition of the viral dsRNA formed during viral replication (Aliyari and Ding, 2009). Small interfering RNAs (siRNAs) directed against protease 2A were shown to inhibit CVB infection in HeLa cells and murine cardiomyocytes (Yuan et al., 2005). siRNA molecules designed to target the CVB viral 2A region successfully reduced viral titers and prolonged survival in susceptible mice (Merl et al., 2005). "
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    ABSTRACT: Coxsackieviruses (CVs) are relatively common viruses associated with a number of serious human diseases, including myocarditis and meningo-encephalitis. These viruses are considered cytolytic yet can persist for extended periods of time within certain host tissues requiring evasion from the host immune response and a greatly reduced rate of replication. A member of Picornaviridae family, CVs have been historically considered non-enveloped viruses - although recent evidence suggest that CV and other picornaviruses hijack host membranes and acquire an envelope. Acquisition of an envelope might provide distinct benefits to CV virions, such as resistance to neutralizing antibodies and efficient nonlytic viral spread. CV exhibits a unique tropism for progenitor cells in the host which may help to explain the susceptibility of the young host to infection and the establishment of chronic disease in adults. CVs have also been shown to exploit autophagy to maximize viral replication and assist in unconventional release from target cells. In this article, we review recent progress in clarifying virus replication and dissemination within the host cell, identifying determinants of tropism, and defining strategies utilized by the virus to evade the host immune response. Also, we will highlight unanswered questions and provide future perspectives regarding the potential mechanisms of CV pathogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Oct 2015 · Virology
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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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    ABSTRACT: Coxsackievirus B3 (CVB3) infection causes myocarditis, pancreatitis, and aseptic meningitis. Targeting antigen-specific T cell reactions might be a promising way to alleviate the inflammatory response induced by CVB3 infection. IL-2-inducible T-cell kinase (ITK), a member of Tec kinase family expressed mainly in T cells, plays an important role in the activation of T cells. The role of ITK in viral myocarditis induced by CVB3 has not been documented. In this study, we inhibited the ITK expression in Jurkat cells, primary human peripheral blood mononuclear cells (PBMC), and mouse splenocytes by ITK-specific siRNA. The inhibition efficiently suppressed cell proliferation (P<0.05) and T-cell related cytokine secretion (P<0.05). In order to inhibit ITK in vivo, the pGCSIL plasmid containing short hairpin RNAs targeting ITK was constructed and transduced into mice infected with CVB3. ITK-inhibited mice showed reduced cell proliferation (3, 5, and 7 days post-challenge, P<0.05) as well as CD4+ and CD8+ T cells (5 days post-challenge, P<0.05). The altered production of inflammatory cytokines alleviated pathologic heart damage and improved mice survival rate (P<0.05). ITK played an important role in the T cell development and represented a new target for the modulation of T-cell-mediated inflammatory response by CVB3 infection.
    Full-text · Article · Jan 2014 · Molecular Immunology
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    • "RNAi-based antiviral therapy has potential to degrade viral RNA and promote viral clearance [22]. In previous publications, RNAi was applied to inhibit coxsackievirus replication by siRNAs targeting viral 2A [23,24], 3D [25], and VP1 [15,25]. These synthetic siRNAs were tansfected into mammalian cells or delivered into mice via the tail vein by high-volume injections and protected cells or organs from virus-mediated injury. "
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    ABSTRACT: Background Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. Methods To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B) expressed by a recombinant vector (pGCL-2B) or a recombinant lentivirus (Lenti-2B) were tansfected in HeLa cells or transduced in mice infected with CVB3. Results ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. Conclusions Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.
    Full-text · Article · Aug 2012 · BMC Infectious Diseases
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