Conserved elements within the genome of foot-and-mouth disease virus; their influence on viral replication

Foot-and-mouth disease (FMD) is caused by a highly contagious virus infection of cloven-hoofed farm animals with high financial importance for countries, such as Denmark, which rely on a significant trade in agricultural products. At present, FMD is exotic to the cloven hoofed livestock in Denmark. However, outbreaks of FMD have previously been detected in other parts of Europe (United Kingdom) during the last decade, thus an introduction of FMD remains a continuing threat to the highly industrialized pig production in Denmark.
Several conserved elements within the genome of the foot-and-mouth disease virus (FMDV) have been identified, e.g. the IRES. Such elements can be crucial for the efficient replication of the genomic RNA. A better understanding of the influence of these elements is required to identify currently unrecognized interactions within the viruses which may be important for the development of anti-viral agents. SHAPE analysis of the entire FMDV genome (Poulsen, 2015) has identified three conserved RNA structures within the coding regions for 2B, 3C and 3D (RNA-dependent RNA polymerase) which might have an important role in virus replication. The FMDV 2A peptide, another conserved element, is responsible for the primary “cleavage” at its own C-terminus (2A/2B junction). It is believed that this “cleavage” is achieved by a protease-independent event termed ribosomal skipping or StopGo, in which the 2A peptide prevents the ribosome from linking the next amino acid to the growing polypeptide. The nature of this “cleavage” has so far not been investigated in the context of the full-length FMDV RNA within cells. The focus of this PhD thesis has been to characterize these elements and their influence on the FMDV replication. In order to fulfil the aims of this thesis a series of studies were performed and the scientific work is presented as three manuscripts.
Manuscript 1 investigated the impact of 2A modifications within the conserved C-terminal D(V/I)E(S/T)NPG↓P motif on FMDV protein synthesis, polyprotein processing and virus viability. Certain amino acid substitutions (E14Q, S15I, S15F and N16H) that have been shown to severely (50-70%) reduce the cleavage activity at the 2A/2B junction compared to the wt, using in vitro assays, have been found to be tolerated within infectious FMDVs. In contrast, substitutions (N16A, P17A, G18A, P19A, and P19G) that inhibit cleavage by 89-100% in vitro, reverted to the wt sequence in the rescued viruses. The 2A substitutions impaired the replication of a FMDV replicon, however surprisingly the viable 2A mutant viruses did not exhibit an attenuation of virus growth in cell culture. Expression of cDNAs encoding a truncated FMDV polyprotein, without any viral proteinases showed that certain amino acid substitutions at residues E14, S15, N16 and P19 resulted in partial “cleavage” indicating that these specific residues are not essential for co-translational “cleavage”. This shows that the StopGo function at the 2A/2B junction is necessary for efficient virus replication. However, maximal cleavage activity does not appear to be essential for the viability of FMDV. Manuscript 2 sought to identify which codons at each position of the coding sequence for the conserved NPG↓P motif at the 2A/2B junction would produce viable progeny viruses. This was achieved using a reverse genetics system with highly degenerate primers with all possible codons present for each of the amino acids individually within the NPG↓P motif. This generated pools of modified plasmids for each codon position from which RNA transcripts were made which were subsequently introduced into cells in culture. The rescued viruses all encoded the NPG↓P motif, confirming the importance of this amino acid sequence. However, at passage two, these four residues were found to be encoded by all possible codons (14 in total) within the rescued pools. Subsequent passages in cell culture revealed a distinct codon bias. Remarkably, this bias matches the codon bias observed within naturally occurring FMDV strains. Interestingly, the codons selected are different for P17 and P19. Residue P17 is preferentially encoded by CCU while P19 is preferentially encoded by CCC. However, a single prolyl-tRNA species recognizes both of these two codons in cattle and pigs, which are the major hosts for FMDV, and suggests a role for the RNA sequence itself. Manuscript 3 examines the influence of three conserved RNA structures within the genome of FMDV on viral protein synthesis and virus viability. Poulsen, (2015) previously identified these RNA structures within the coding regions for the FMDV 2B, 3C and 3D proteins using SHAPE probing. Interestingly, the structures had significant lower synonymous substitution rates compared to the remainder of the genome. However, introduction of synonymous substitutions which disrupted the structures but did not alter the amino acid sequence are tolerated and were retained after three passages in cell culture. The same substitutions did not impair the replication of a FMDV replicon either. This shows that the identified structures located in the coding region of 2B, 3C and 3D are not required for FMDV replication in BHK