Identification of plasticity and interactions of a highly conserved motif within a picornavirus capsid precursor required for virus infectivity

Figures

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  Figure 1. Picornavirus capsid assembly. (a) The genome organization of FMDV is shown. The viral RNA encodes a large polyprotein that is processed to yield precursors and finally mature products. The FMDV capsid protein precursor (P1-2A) includes the conserved YCPRP motif near the C-terminus of VP1. The Leader protease (Lpro) cleaves itself from the P1-2A precursor. Ribosomal skipping at the C-terminus of 2A is marked with an arrow. The 3Cpro (red) is responsible for cleavage of all other junctions. (b) A logoplot showing variation in this conserved motif amongst a collection of picornavirus sequences (from http://www.virology.wisc.edu/acp/Aligns/aligns/picorna.p1) was generated using https://weblogo.berkeley.edu. (c) The pathway of capsid assembly is shown from the processing of the capsid precursor P1-2A by the 3Cpro, to the formation of protomers, the assembly of pentamers and finally the generation of complete capsids. The cleavage of VP0 to VP4 and VP2 occurs during the formation of virus particles. The structures of the pentamer and virus particle are from PDB 4GH421.

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  Figure 2. The plasticity of the YCPRP motif within infectious FMDV. Mutagenesis was performed on defective mutant full-length FMDV cDNA templates (see text) to modify the codon for each residue within the YCPRP individually to all possible codons (NNN). The pools of plasmids obtained were sequenced and the heterogeneity in each pool is shown. RNA transcripts were prepared from each mutant pool separately and introduced into cells to recover infectious virus. CPE was observed at passage 1 (c.f. the mutant template) in each case and then amplified through 2 passages in total. RNA was extracted from these rescued viruses, converted to cDNA and sequenced throughout the P1-2A coding region. No secondary substitutions were observed and heterogeneity was only observed within the sequence encoding the conserved motif in VP1. From each cDNA pool, a cDNA fragment, encoding VP1-2A, was amplified and inserted into a TOPO vector and individual clones (10–33) were sequenced. Representatives of the distinct individual sequences are shown together with the encoded amino acid sequence. The number and frequency (%) of each codon within the individual clones analyzed is shown. Note, within the rescued…

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  Figure 3. A second site substitution within VP2 compensates for the deleterious effect of the VP1 R188A modification in the FMDV P1-2A capsid precursor. Plasmids encoding wt or mutant forms of the FMDV P1-2A (as indicated) were expressed alone (odd numbered lanes) or with the FMDV 3Cpro (even numbered lanes) within BHK cells, as described in Methods section. Cell lysates were analyzed by SDS-PAGE and immunoblotting using guinea pig anti-FMDV O-Man antisera. Bound antibodies were visualized using the anti-guinea pig HRP-conjugated secondary antibodies and a chemiluminescence detection kit. The unprocessed P1-2A precursor and the VP0 processing product are indicated. Note the enhanced processing of the P1-2A (VP1 R188A) mutant in the presence of the substitution VP2 (W129R).

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  Figure 4. Location of residues VP1 R188 (within the conserved motif) and VP2 W129 in the virus structure. (a) The structure of a pentamer from within the FMDV A22 Iraq capsid is shown (from PDB 4GH421). (b) An expanded view of a single protomer viewed (from a different angle) is shown and the box marks a further expanded view, as shown in (c) with the residues VP1 R188 and VP2 W129 at the interface between VP1 and VP2 indicated. VP1 is shown in dark blue and VP2 is in light blue.

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  Figure 5. Conservation of VP2 sequences among picornaviruses. The amino acid sequence upstream of residue W129 within VP2 of FMDV (marked with an ↓) is highly conserved among other FMDVs and also among other picornaviruses. Amino acids that are identical among most FMDVs and are also conserved in other virus genera are highlighted in yellow. Non-conserved residues that have similar properties to the conserved residues are highlighted in other colours (see key). Residues that differ from FMDV but are highly conserved within particular virus genera or species are also marked. The selected viruses were derived from alignments available at (http://www.virology.wisc.edu/acp/Aligns/aligns/picorna.p1).