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

• Published in: Journal of general virology Vol. 104; no. 7
• Main Authors: Dobson, Samuel J, Ward, Joseph C, Herod, Morgan R, Rowlands, David J, Stonehouse, Nicola J
• Format: Journal Article
• Language: English
• Published: England 01.07.2023
• Subjects: Animals; Foot-and-Mouth Disease; Foot-and-Mouth Disease Virus - genetics; Foot-and-Mouth Disease Virus - metabolism; Picornaviridae - genetics; RNA, Viral - metabolism; Virus Replication - genetics; replication; FMDV; foot-and-mouth disease virus; negative sense; strand-specific assay; cre
• ISSN: 0022-1317; 1465-2099; 1465-2099
• DOI: 10.1099/jgv.0.001871

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus within the 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, but 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 quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled 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 quantitative PCR, 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 -acting replication elements and provide direct evidence for their roles in negative-strand synthesis.