Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at distal sites

Figures

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  Fig 1. Schematic representation of the polyprotein processing in three different genera of picornaviruses. a) The RNA genome of viruses in the genus Aphthovirus, in which FMDV is the prototypic member, encodes a polyprotein that is processed into four primary products namely the Leader protease (Lpro), P1-2A, P2 and P3 during, and after, synthesis. The “cleavage” at the 2A/2B junction occurs during synthesis by a mechanism known as “ribosomal skipping” [8] or “StopGo” [9]. The Lpro mediated cleavage occurs at its own C-terminus, i.e. at the L/P1-2A junction while the 3Cpro is responsible for cleavage of the P2/P3 junction. The Cardiovirus polyprotein is also processed into the primary products, Leader, P1-2A, P2 and P3 but the Leader protein is removed from the capsid precursor by the 3Cpro. The Entero- and Rhinoviruses, do not have a Leader protein and the polyprotein is thus processed into only three primary products, namely P1, P2 and P3. In the Entero- and Rhinoviruses the 2A protein is a protease that cleaves the P1/P2 junction. The 3Cpro is responsible for cleavage of the P2/P3 junction. b) The 3Cpro is sufficient for processing of the capsid precursor, P1-2A, in Aphthoviruses and…

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  Fig 2. Truncations in the C-terminus of FMDV VP1 affect capsid precursor processing. a) Constructs encoding the FMDV P1-2A (wt) were modified to introduce truncations at different sites in the VP1 coding sequence. The 2A L1Stop truncation served as a second positive control for processing. b) Cell lysates from BHK cells (infected with vTF7-3 [45]) transfected with plasmids that express the FMDV P1-2A (wt or truncated at the VP1) alone (odd numbered lanes) or with the plasmid pSKRH3C [46] encoding the FMDV 3Cpro (even numbered lanes) were analyzed by immunoblotting. Molecular mass markers (kDa) are indicated on the left. A negative control (No DNA) was included (lane 17). The different structural proteins are indicated on the right of the figure and were detected 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.

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  Fig 3. A short region near the C-terminus of FMDV VP1 is required for capsid precursor processing Cell lysates were prepared from BHK cells (infected with vTF7-3 [45]) following transfection with plasmids that express the FMDV P1-2A wt or with small deletions as indicated either alone (odd numbered lanes) or with the plasmid pSKRH3C [46] encoding the 3Cpro (even numbered lanes). The samples were analyzed by immunoblotting. A positive control with a deletion known to be tolerated in infectious FMDV [20] was included, P1-2A (VP1 Δ142–154), lanes 13 and 14. Molecular mass markers (kDa) are indicated on the left. A negative control (No DNA) was used in lane 15. The structural proteins corresponding to the different bands are indicated on the right of the figure and were detected 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.

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  Fig 4. A region near the C-terminus of FMDV VP1 required for processing of the VP3/VP1 junction. a) The P1-2A precursor (wt or with small deletions as indicated) was expressed alone or with 3Cpro in transient expression assays in BHK cells as for Fig 3. Cell lysates were prepared and analyzed by immunoblotting. The structural proteins corresponding to the different bands are indicated and were detected using guinea pig anti-FMDV A-Iraq antisera. Bound antibodies were visualized using the anti-guinea pig HRP-conjugated secondary antibodies and chemiluminescence detection. The odd numbered lanes show the P1-2A precursors expressed alone and the even numbered lanes show the precursor co-expressed with 3Cpro. Molecular mass markers (kDa) are indicated on the left. A negative control (No DNA) is included in lane 9. A positive control with a deletion known to be tolerated in replicating FMDV [20] was included, P1-2A (VP1 Δ142–154), lanes 7 and 8. b) The 2A L2P substitution was introduced into the plasmids encoding P1-2A (wt), P1-2A (VP1 Δ191–195), P1-2A (VP1 Δ194–199) and the positive control P1-2A (VP1 Δ142–154). This 2A L2P substitution blocks cleavage of the VP1/2A junction and increased…

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  Fig 5. Identification of amino acids between residues VP1 185 and 188 required for capsid precursor processing The P1-2A precursors (wt or having alanine substitutions between VP1 185 and VP1 188) were expressed alone or in the presence of 3Cpro and analyzed by immunoblotting (as above). The structural proteins corresponding to the different products are indicated on the right of the figure and were detected using guinea pig anti-FMDV O-Man antisera (a) or guinea pig anti-FMDV A-Iraq antisera (b). Bound antibodies were visualized using the anti-guinea pig HRP-conjugated secondary antibodies and a chemiluminescence detection kit. Molecular mass markers (kDa) are indicated on the left. A negative control (No DNA) is included in lane 11.

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  Fig 6. Identification of residues between VP1 189 and 193 required for processing at the VP3/VP1 junction. The P1-2A precursors (wt or with alanine substitutions between VP1 189 and VP1 193, with the exception of VP1 192 where an alanine were substituted to a serine, as indicated) were expressed alone or in the presence of 3Cpro and analyzed by immunoblotting. The capsid proteins are indicated on the right of the figure. Proteins were detected using guinea pig anti-FMDV O-Man antisera (a) or with guinea pig anti-FMDV A-Iraq antisera (b). Bound antibodies were visualized using the anti-guinea pig HRP-conjugated secondary antibodies and a chemiluminescence detection kit. Molecular mass markers (kDa) are indicated on the left. A negative control (No DNA) is included in lane 13.

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  Fig 7. Alignment of the VP1 C-terminus from different FMDV serotypes Amino acid alignment of the VP1 C-terminus of different strains of FMDV, including examples of all seven serotypes. The yellow color represents amino acids that are more that 97% conserved among the different strains. The conserved motif YCPRP (VP1 185–189) is marked with a box in the consensus sequence. The alignment was prepared from sequences published previously [15].

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  Fig 8. Alignment of different picornaviruses at the VP1 C-terminus Amino acid alignment of the VP1 C-terminus of diverse picornaviruses are shown. The black box indicates the highly conserved motif (VP1 185–189 in FMDV) that is important for correct processing of the capsid precursor. The sequences are numbered according to the FMDV residues. The yellow color represents amino acids that are identical with the YCPRP motif found in FMDV. Conservative substitutions are indicated with a similar color i.e. yellow for Y, orange for F and pink for W. The C residue at VP1 186 (according to FMDV VP1 numbers) is conserved in 31 out of 48 aligned picornaviruses (marked with yellow). The P and R at VP1 187 and 188 are 100% conserved amongst all of the aligned picornaviruses (marked with yellow). The P at VP1 189 (according to FMDV VP1 numbers) is conserved in 35 out of 48 of the aligned picornaviruses, differences from the P are seen in all of the hepatoviruses and a few of the enteroviruses. The alignment was taken from Ann Palmenberg’s laboratory website (http://www.virology.wisc.edu/acp/Aligns/aligns/picorna.p1) and highlighted manually. Abbreviations: FMDV = Foot-and-mouth disease virus, EMC =…

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  Fig 9. Model: Potential role of chaperone binding to a conserved motif for correct capsid precursor folding a) Hsp90 (or other chaperones and co-chaperones) is believed to bind to the P1-2A precursor (or P1 for enteroviruses) to facilitate folding into a state where the 3Cpro (or 3CDpro) is able to process the precursor to the mature structural capsid proteins prior to particle assembly. b) It is hypothesized that the conserved motif, YCPRP, in the C-terminus of VP1 is required for the binding of a chaperone (or co-chaperone). In the absence of this interaction, the precursor will be incorrectly folded and hence 3Cpro (or 3CDpro) will not recognize the cleavage sites; the misfolded, intact, precursor will be degraded by the proteasome.