The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

Abstract

word count: 194 22 Text word count: 5168 23 24 25 author/funder. All rights reserved. No reuse allowed without permission. Abstract 27 The positive stranded RNA genomes of picornaviruses comprise a single large open reading 28 frame flanked by 5′ and 3′ untranslated regions (UTRs). Foot-and-mouth disease virus (FMDV) 29 has an unusually large 5′ UTR (1.3 kb) containing five structural domains. These include the 30 internal ribosome entry site (IRES), which facilitates initiation of translation, and the cis-acting 31 replication element (cre). Less well characterised structures are a 5′ terminal 360 nucleotide 32 stem-loop, a variable length poly-C-tract of approximately 100-200 nucleotides and a series of 33 two to four tandemly repeated pseudoknots (PKs). We investigated the structures of the PKs 34 by selective 2′ hydroxyl acetylation analysed by primer extension (SHAPE) analysis and 35 determined their contribution to genome replication by mutation and deletion experiments. 36 SHAPE and mutation experiments confirmed the importance of the previously predicted PK 37 structures for their function. Deletion experiments showed that although PKs are not essential 38for replication, they provide genomes with a competitive advantage. However, although 39 replicons and full-length genomes lacking all PKs were replication competent, no infectious 40 virus was rescued from genomes containing less than one PK copy. This is consistent with our 41 earlier report describing the presence of putative packaging signals in the PK region. 42 43 author/funder. All rights reserved. No reuse allowed without permission.

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

“…The construction, linearisation and purification of FMDV replicon plasmids has been described previously (24, 60). Linear plasmids were transcribed in vitro using the T7 RiboMAX Express Large Scale RNA Production System (Promega) using ‘half sized’ reactions whereby 250 ng linear plasmid was added to a reaction mixture using half the volume suggested for all reagents in the manufacturer’s protocol.…”

“…The single viral open reading frame (ORF) is flanked by untranslated regions (UTRs) at the 5' and 3' ends of the genome. The FMDV 5' UTR is longest amongst known picornaviruses and contains several highly structured domains that are essential to the virus lifecycle (24)(25)(26)(27)(28)(29)(30). The S fragment is a ~360 nucleotide hairpin-loop that occupies the same location (at the extreme 5' end of the 5' UTR) as the cloverleaf found in enteroviruses (31)(32)(33)(34).…”

“…It appears to be involved in immune modulation and viral replication via interactions with host and viral proteins (27,35,36). Adjacent to the S-fragment is a poly-C sequence of unknown function, followed by two to four RNA pseudoknots, which appear to be involved in assembly of infectious virions in addition to genome replication (24,(37)(38)(39). Adjacent to the pseudoknots is a small hairpin-loop known as the cis-acting replication element (cre) that in other picornaviruses is located within the ORF (25,40).…”

Mutagenesis Mapping of RNA Structures within the Foot-and-Mouth Disease Virus Genome Reveals Functional Elements Localised in the Polymerase (3Dpol) Encoding Region

“…Elsewhere in the 5' UTR, the presence of multiple (2-4) pseudoknots downstream of the poly(C) tract has been shown to determine virus tropism (41,46,47). Other key and well-characterised RNA structural elements include a type II internal ribosome entry site (IRES), which initiates capindependent translation of the viral genome (41,(48)(49)(50)(51); while the cis-acting replication element (cre) acts as a template for uridylylation of the VPg (3B) protein, which then acts as a primer for synthesis of viral RNA (52,53).…”

“…Within the 5’ UTR of the FMDV genome, the S-fragment forms a single, long hairpin structure (293-381 nucleotides (nts) in length) and has been reported to play a role in viral replication and innate immune modulation (41-45). Elsewhere in the 5’ UTR, the presence of multiple (2-4) pseudoknots downstream of the poly(C) tract has been shown to determine virus tropism (41, 46, 47). Other key and well-characterised RNA structural elements include a type II internal ribosome entry site (IRES), which initiates cap-independent translation of the viral genome (41, 48-51); while the cis -acting replication element ( cre ) acts as a template for uridylylation of the VPg (3B) protein, which then acts as a primer for synthesis of viral RNA (52, 53).…”