The siRNA with AHCe-attenuating potential identified in this study had a sequence complementary to the gene encoding the 3D RNA-dependent RNA polymerase, which has a highly conserved sequence.
14 Because of the lack of proofreading ability of various virus RNA polymerases, even supposedly single species of particular RNA viruses really consist of collections of quasi-species, with genomic diversity and genetic instability, in nature.
36,37 Targeting of highly conserved regions is thus essential for the development of siRNA-based therapeutic agents inhibiting the replication of RNA viruses. For example, an siRNA-targeting HEB, including CVB3, could be developed after study of the 3D region.
11,22 Genes encoding other nonstructural proteins, such as 2A and 2C, have been identified as promising alternatives for RNA targeting.
24,38 The
cis-acting element (CRE) region within 2C is a particularly promising target for the design of potent anti-HEB siRNAs, inhibiting replication of viruses within the enterovirus B subgroup.
38 The CRE region, a core
cis-acting element essential for virus genome amplification, is extremely sensitive to genetic alteration.
39 In addition, the CRE region spatially forms a large loop, allowing easy access to the virus replication machinery and thus to siRNA.
38,39 Indeed, CVA24-specific siRNA targeting the CRE region showed high-level anti-CVA24 activity against clinically isolated CVA24s of diverse sequences.
26 Nevertheless, an siRNA targeting the CRE region was not selected in this work, in which we targeted CVA24 and EV70 viruses, which belong to different enterovirus subgroups (HEC and HED). Close examination of sequences in the CRE region revealed multiple mismatches between HEC and HED. Two sequence mismatches were located at positions 10 and 11 from the 5′ end of CAPSID-suggested siRNA sequences. Because actual cleavage of target RNA occurs between these positions, absolute sequence identity of target RNA and siRNA is required.
33 Recombination among enterovirus genomes is a relatively frequent phenomenon, primarily at the noncoding region of the EV genomes, including 2C and the highly conserved 3D region. Therefore, there is still a chance that some specific siRNAs fail to inhibit the viral replication.
38