Methods for detection and study of virus-derived small RNAs produced from the intramolecular base-pairing region of the picornavirus genome

There is conclusive evidential support for the existence of virus-derived small RNA (vsRNA) in mammals. Two types of vsRNA have been reported from picornaviruses. The first is virus-derived short-interfering RNA (vsiRNA) that is processed from viral double-stranded RNA intermediates during RNA replication. The other is small RNA derived from the highly base-paired single-stranded genomic region, e.g. the internal ribosome entry site (IRES) of picornaviruses. vsiRNA interacts with the Argonaute protein to control viral RNA replication through the process of RNA interference. However, the function of structure-based vsRNA is largely unknown. We previously identified vsRNA1 generated from the enterovirus-A71 (EV-A71) IRES region by the endogenous enzyme Dicer. Exogenous vsRNA1 can inhibit IRES activity both in vivo and in vitro, hence viral replication is inhibited. Here we describe key methods used to characterize vsRNA, including annotation by next-generation sequencing, abundance measurement by Northern blotting, determination of Dicer-dependence by gel-shift assay and in vitro cleavage assay, and the inhibitory effect on IRES activity via in vitro translation assay.