Insights into Polyprotein Processing and RNA-Protein Interactions in Foot-and-Mouth Disease Virus Genome Replication

Figures

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  FIG 1. A single amino acid substitution at the 3B3-3C junction prevents FMDV replicon replication. (A) Schematic diagram of the FMDV replicon. (B) Sequence alignments of the 3B cleavage junctions with the 3B1,2,3Y3F, 3B3/2, and 3B3T>K mutants. (C) Replication of replicons containing 3B3 mutations as well as the WT ptGFP replicon (ptGFP) and replication-defective controls containing inactivating mutations in 3Dpol (3DGNN), or 3B proteins (3B1,2,3Y3F). GFP expression was monitored hourly for 24 h. The graph shows GFP positive cells per well at 8 h posttransfection when replication is maximal. Significance compared to WT control (n = 3 ± SEM; * = P ≤ 0.05).

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  FIG 2. A single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis and drives the production of novel precursors. (A) Plasmids expressing the WT or mutant 3B3T>K FMDV polyprotein precursors were used to prime [35S] labeled pulse-chase in vitro coupled transcription/translation reactions. At regular intervals, samples were taken and stopped by the addition of 2× Laemmli buffer. Proteins were separated by SDS-PAGE and visualized by autoradiography. The identity of some FMDV proteins is shown. (B) The percentage of protein or protein precursor was quantified as a total percentage of 2C or 3Dpol containing products, as appropriate (n = 2 ± SD). (C) Duplicate reactions were incubated for 90 min before immunoprecipitation of 2C containing precursors with anti-2C antibodies. The pre- and post precipitation samples were separated by SDS-PAGE and visualized by autoradiography. Arrows show the identity of 2C containing proteins, based on predicted molecular weights. F/T refers to the flow through samples.

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  FIG 3. The 3B3T>K amino acid substitution drives trans-mediated precursor proteolysis. Plasmids expressing proteolytically inactive polyproteins with or without the 3B3T>K substitution (termed 3B3T>K, 3CC163A, and 3CC163A, respectively) were used to prime coupled [35S] labeled transcription/translation assays, followed by unlabeled amino acid chase. To a duplicate set of reactions 10 μM purified 3Cpro was added (+3C) immediately after chase. As controls, reactions were set up alongside the WT or 3B3T>K polyproteins without an inactivated 3Cpro. At regular intervals, samples were taken and reactions stopped by the addition of 2× Laemmli buffer. (A) Proteins were separated by SDS-PAGE and visualized by autoradiography. The relative proportion of uncleaved 3AB1,2,3CD (B) or 3CD (C) was quantified as a percentage of 3Dpol containing products (n = 2 ± SD).

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  FIG 4. The 3B3T>K amino acid substitution drives trans-mediated precursor proteolysis. Plasmids expressing proteolytically inactive versions of WT or 3B3T>K polyproteins (3CC163A and 3B3T>K3CC163A, respectively) were used to prime coupled [35S] labeled transcription/translation assays in the presence of 3AB1,2,3CproD, a proteolytically active precursor with all cleavage boundaries mutated to prevent self-proteolysis (+3AB123CproD). At regular intervals, samples were taken and reactions stopped by the addition of 2× Laemmli buffer. (A) Proteins were separated by SDS-PAGE and visualized by autoradiography. The relative proportion of uncleaved 3AB1,2,3CD (B) or 3CD (C) was quantified as a percentage of 3Dpol containing products (n = 2 ± SD).

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  FIG 5. The 3B3T>K substitution prevents complementation of 2C mutants in trans. (A) Schematic of the trans-complementation experiment which involved co-transfecting BHK-21 cells with mCherry replicons containing replication-defective 2C or 3B mutations together with a WT ptGFP, ptGFP-3B3T>K or ptGFP-3DGNN replicon. Fluorescent protein expression was monitored hourly for 24 h. The data show (B) ptGFP positive cells per well or (C) mCherry positive cells per well at 8 h posttransfection (n = 2 ± SD).

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  FIG 6. S-fragment deletions allow trans-complementation of cis-acting replication components. (A) Schematic of the trans-complementation experiment which involved co-transfecting BHK-21 cells with mCherry replicons containing S-fragment deletions together with a WT ptGFP, ptGFP-3B3T>K or ptGFP-3DGNN replicon. Fluorescent protein expression was monitored hourly for 24 h. The data show (B) ptGFP positive cells per well or (C) mCherry positive cells per well at 8 h posttransfection (n = 2 ± SD).

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  FIG 7. Detection of FMDV RNA-protein complexes by PLA. (A) BHK-21 cells were co-transfected with BrU-labeled ptGFP replicon RNA together with 3DHA-labeled replicon RNA. At 4 h posttransfection cells were fixed and 3DHA-BrU RNA complexes detected by proximity ligation assay (PLA) using anti-HA and anti-BrU primary antibodies together with PLA-labeled secondary antibodies. The in-situ PLA signal is detected as foci in the cell cytoplasm (pseudo-colored magenta). Cell nuclei were stained with DAPI (pseudo-colored blue). Images were captured on a Zeiss LSM-880 confocal microscope (bar 10 μm). (B) The mean number of individual PLA foci per cell were quantified from an average of at least 20 GFP positive cells per condition spread across two biological replicate experiments (n = 2 ± SD).

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  FIG 8. Model for regulation of FMDV replication by polyprotein processing. The 3B3T>K substitution drives rapid processing preferentially down one pathway, as demonstrated by the reduced levels of of 2C and 3Dpol with increased formation of 3AB1,2,3 and 3CD. To support replication of the 3B3T>K replicon in trans, the entire non structural polyprotein is required to deliver a complex of functional components including 2C and 3Dpol, as demonstrated by the wild-type processing schematic. This likely provides products from alternative cleavage pathways such as 3B1,2,3 and 3CD.