Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways

Abstract

The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from "helper" replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the "helper" replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy.

Fig 1. Residues at the 3B-3C boundary are important for replicon replication.

(A) Schematic of the FMDV sub-genomic replicons bearing 3B deletions and tyrosine mutations used in this study. The 3B region is expanded for clarity. (B) BHK-21 cells seeded into 24 well plates were transfected with mCherry replicons bearing 3B deletions or mutations as well as wild-type (wt) and replication-defective polymerase mutant (3D^pol-GNN) controls. Transfections were performed with replicons from which 3B1, 3B2 or 3B3 had been deleted (Δ3B1, Δ3B2, and Δ3B3, respectively), both 3B1 and 3B2 deleted in tandem (Δ3B1+2) or point-mutation to the uridylatable tyrosine of 3B1, 3B2 or 3B3 (3B1^Y3F, 3B2^Y3F or 3B3^Y3F, respectively). Expression of mCherry was monitored hourly over a 24 hour period. Data shown represent mean mCherry positive cells per well at 8 hours post-transfection. Significance compared to wild-type control (n = 3 ± SD, * = p<0.05, ** = p<0.01). (C) Alignment of the replicon 3B amino acid sequences and the chimeric 3B boundary mutations (3B2/3 and 3B3/2) showing the specific amino acid sequences of each mutation underlined. (D) BHK-21 cells seeded into 24 well plates were transfected with a ptGFP replicon with the positions of 3B2 and 3B3 exchanged (3B132) and expression of ptGFP monitored hourly over a 24 hour period. The wild-type, 3D^pol-GNN, Δ3B3 and 3B123^Y3F constructs were included as controls. Data shown represent mean ptGFP positive cells per well at 8 hours post-transfection. (E) BHK-21 cells seeded into 24 well plates were transfected with mCherry replicons bearing chimeric 3B mutations and expression of mCherry monitored hourly over a 24 hour period. The wild-type, 3D^pol-GNN, Δ3B1+2 and Δ3B3 constructs were included as controls. Data shown represent mean mCherry positive cells per well at 8 hours post-transfection. Significance compared to wild-type control (n = 3 ± SD, * = p<0.05, ** = p<0.01).

Fig 2. Mutations at the 3B-3C boundary disrupt P3 polyprotein processing.

Plasmid constructs expressing wild-type FMDV P3 or the 3B3/2 chimeric mutant polyprotein were used to assemble coupled transcription/translation reactions with [³⁵S] labelled methionine. Reactions were incubated for 40 minutes before addition of excess unlabelled methionine/cysteine, samples were taken at regular intervals and reaction stopped by the addition of 2 x Laemmli buffer. (A) Proteins were separated on 12% SDS-PAGE and visualised by autoradiography. The positions of FMDV protein products are indicated by arrows, the 3D* product represents a degradation or cleavage product of 3D^pol (as confirmed by Western blot, see S2 Fig). Control reactions were assembled using empty expression vector alone. The proportion of uncleaved P3 (B) and 3D^pol (C) product was quantified as a percentage of the total 3D^pol containing products (n = 2 ± SD).

Fig 3. Mutations at the C-terminus of 3B3 prevent complementation of 3D^pol mutations but not 3B mutations in trans.

(A) BHK-21 cells seeded into 24-well plates were co-transfected with mCherry replicons bearing replication-defective 3B or 3D^pol mutations or controls and wild-type ptGFP, ptGFP-3B123^Y3F or ptGFP-3D^pol-GNN replicon. In (A) all the mCherry replicons contained a full IRES (+). In (B) the mCherry replicons contained a deletion of the entire IRES (Δ), in addition to the indicated non-structural protein mutation (3D^pol-GNN, 3B123^Y3F, 3B3/2). Co-transfections were performed with yeast tRNA (bars labelled ‘na’) as a negative control (i.e. no complementation) in both experiments. Expression of ptGFP is shown representing mean positive cells per well at 8 hours post transfection (n = 3, ± SD). For clarity the statistics were excluded from the figure. The mCherry data is shown in S3 Fig. In (C), after the initial the co-transfection, total RNA was harvested and replicons RNA purified by oligo(dT) precipitation. 100 ng of total oligo(dT) purified RNA was re-transfected into BHK-21 cells seeded into 24-well plates and expression of ptGFP and mCherry were monitored hourly. Data shows mean ptGFP positive cells per well at 8 hours post transfection. Significance compared to plus yeast tRNA negative control. (n = 5 ± SD, * = p<0.05, ** = p<0.01).

Fig 4. O1 Kaufbeuren virus containing Δ3B3 mutation changes 3B2-3C cleavage site to resemble 3B3-3C boundary.

(A) Δ3B1+2, Δ3B3, 3B2/3 and 3B3/2 replicons were converted into infectious copy (IC) constructs by insertion of the capsid encoding region of O1 Kaufbeuren (O1K) FMDV isolate. RNA transcribed from IC constructs was transfected into BHK-21 cells and virus was recovered by freeze/thawing. Recovered viruses were blindly passaged up to four times in BHK-21 cells (BHK p1-4); each time infected cells were monitored for appearance of CPE. (B) All four viruses which had undergone four passages (BHK p4) were sequenced using Illumina MiSeq platform. Consensus and subconsensus sequences of recovered viruses (BHK p4) were compared to the input IC construct (input_IC). Part of sequences (nucleotide in black and amino acid in dark grey) where changes in recovered viruses were observed in comparison to input IC construct are shown. Mutated nucleotides/amino acids are underlined; percentage of virus population bearing the mutation is shown underneath in brackets. Dashed line indicates the scissile bond dipeptide cleavage site with the flanking boundary residues indicated and designated P5 to P5' and according to the standard nomenclature. Nucleotide range shown in brackets indicates position of the presented sequence in relation to genome of recovered viruses.

Fig 5. A single reversion mutation at the 3B-3C boundary restores replicon replication.

BHK-21 cells seeded into 24 well plates were transfected with mCherry replicons bearing 3B mutations as indicated, alongside wild-type (wt) and replication-defective polymerase mutant (3D^pol-GNN) controls, and expression of mCherry monitored hourly over a 24 hour period. Data shown represents mean mCherry positive cells per well at 8 hours post-transfection. Significance compared to wild-type control (n = 3 ± SD, * = p<0.05, ** = p<0.01).

Fig 6. Reversion mutations at the 3B-3C boundary restore P3 polyprotein processing.

Plasmid constructs expressing wild-type or mutant P3 polyprotein were used to assemble coupled transcription/translation reactions with [³⁵S] labelled methionine. Reactions were incubated for 40 minutes before addition of excess unlabelled methionine/cysteine, samples taken at regular intervals and reactions stopped by the addition of 2 x Laemmli buffer. (A) Proteins were separated on 12% SDS-PAGE and visualised by autoradiography. The positions of FMDV protein products are indicated by arrows. Control reactions were assembled using empty expression vector alone. The proportion of uncleaved P3 (B) and 3D^pol (C) product was quantified as a percentage of the total 3D^pol containing products (n = 2 ± SD).