Coxsackievirus B3 (CVB-3) belongs to the Picornaviridae family of enterovirus genus of pathogens that cause a great number of human diseases. A viral infection is associated with many pathological states such as: myocarditis, dilated cardiomyopathy, pericarditis, pleurodynia, systemic infection in infants, aseptic meningitis, and pancreatitis. Since viral diseases, especially in their chronic state, are difficult to treat, there has not been as yet, any specific therapeutic developed against coxsackievirus till date. CVB-3 is a single stranded, positive-sense RNA virus that encodes one large open reading frame flanked by two untranslated regions (UTR). The 5NUTR contains an IRES element that directs the translation process and a cloverleaf structure that regulate viral replication. The complementary, 3N terminal region of the replicative strand is also believed to be crucial for the replication of events. The secondary structure RNA elements regulate the most important processes in the viral propagation cycle. The mechanisms that rule the CBV-3 gene expression, its genome structure and the key steps of its viral life cycle are being reviewed in the hope that better knowledge of these processes will lead to better understanding of the molecular biology of CVB-3 and to the design of an effective therapy against this enterovirus.

No abstract

RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5’UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.