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.

Emergency Services of Viral RNAs: Repair and Remodeling

“…The indels may not be strictly neutral, but if they are associated with some fitness cost, the detrimental phenotypic changes are likely to be suppressed by some secondsite mutations (see below). In some cases, such indels appeared to even be advantageous, judging by their strong conservation in representatives of a given picornavirus species, as is the case, for example, with the triplication of the VPg gene in the FMDV RNA (276,277) and the duplication of the 5=-terminal cis-element of the bovine enterovirus RNA (278,279). Conserved duplications in untranslated and coding RNA regions of other viruses have been described as well (cf.…”

The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

“…Replication was monitored by measuring ptGFP reporter expression, in parallel with transfection of a wt and 3D-GNN replicon, where the 3D-GNN replicon is used to monitor ptGFP expression resulting from translation of input RNA in the absence of replication. Reporter expression was recorded using an IncuCyte Zoom automatic fluorescent microscope and is shown at 8 hours post-transfection, analysed by our standard methods (8, 10, 38).…”

“…BHK-21 cells were transfected in 25 cm 2 flasks with 8 µg per flask of infectious clone-derived RNA using TransIT transfection reagent (Mirus) as described previously (32). 24 hours post-transfection cell lysates were freeze-thawed and clarified by centrifugation.…”

“…Recovered viruses at passage 4, were sequenced using an Illumina Miseq (illumine) using a modified version of a previously described PCR-free protocol ((32, 33)). Total RNA was extracted from clarified passage 4 lysates using TRizol reagent (Thermo Fisher Scientific) and residual genomic DNA removed using DNA-free DNA removal Kit (Thermo Fisher Scientific).…”

“…The P1 region encodes the capsid structural proteins VP1, VP3, and VP0 (which is further processed to VP2 and VP4 during virus assembly) (6). The P2 and P3 regions encode the non-structural proteins 2B and 2C and 3A, 3B (1-3) (VPg), 3C pro and 3D pol respectively (7, 8). Due to disease security restrictions, work with infectious virus is limited to a few high containment facilities.…”