Highly structured genomic sequences mediate the function of a RNA virus polymerase

Abstract Secondary and tertiary RNA structures play key roles in genome replication of single stranded positive sense RNA viruses. Complex, functional structures are particularly abundant in the untranslated regions of picornaviruses, where they are involved in initiation of translation, priming of new strand synthesis and genome circularisation. The 5′ UTR of foot-and-mouth disease virus (FMDV) is predicted to include a c. 360 nucleotide-long stem-loop, termed the short (S) fragment. This structure is highly conserved and essential for viral replication, but the precise function(s) are unclear. Here, we used selective 2′ hydroxyl acetylation analysed by primer extension (SHAPE) to experimentally-determine aspects of the structure, alongside comparative genomic analyses to confirm structure conservation from a wide range of field isolates. To examine its role in virus replication, we introduced a series of deletions to the distal and proximal regions of the stem loop. These truncations affected genome replication in a size-dependent and, in some cases, host cell-dependent manner. Furthermore, during passage of viruses incorporating the largest tolerated deletion from the proximal region of the S fragment stem loop, an additional mutation was selected in the viral RNA-dependent RNA polymerase, 3D pol , which influenced the function of the enzyme. These data suggest that the S fragment and 3D pol interact in the formation of the FMDV replication complex.