A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis -acting elements in foot-and-mouth disease virus replication

Foot-and-mouth-disease virus (FMDV), the etiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the Picornavirus family. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, however, the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as qPCR preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific qPCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis -acting replication elements and provide direct evidence for their roles in negative-strand synthesis.