Abstract
BackgroundSuccess of a viral infection requires that each infected cell delivers a sufficient number of infectious particles to allow new rounds of infection. In picornaviruses, viral replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown.Methodology and Principal FindingsWe have constructed four mutant FMDVs that encode only one VPg: either VPg1, VPg3, or two chimeric versions containing part of VPg1 and VPg3. All mutants, except that encoding only VPg1, were replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21 cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with restoration of plaque formation. Specifically, replacement R55W in 2C was repeatedly found in several mutant viruses that had regained competence in plaque development. The effect of R55W in 2C was to mediate an increase in the extracellular viral RNA release without a detectable increase of total viral RNA that correlated with an enhanced capacity to alter and detach BHK-21 cells from the monolayer, the first stage of cell killing.ConclusionsThe results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. Implications for picornaviruses pathogenesis are discussed.
Highlights
Contrary to initiation of cellular DNA replication which is primed by RNA molecules synthesised by cellular primases [1], viruses use a wide variety of molecular mechanisms to initiate genome replication, that include de novo initiation, priming by proteins or by self generated 39–ends of templates, and ‘cap–snatching’, among other mechanisms [2]
The results show that a complete sequence of VPg3 is not essential for viral replication, but VPg1–containing mutants require additional residues from VPg3 in their C–terminus for replication
One is the multifunctional nature of many proteins encoded in the compact RNA genomes, in the case of picornaviruses in which several intermediates obtained in the processing of the polyprotein play essential roles in the virus life cycle
Summary
Contrary to initiation of cellular DNA replication which is primed by RNA molecules synthesised by cellular primases [1], viruses use a wide variety of molecular mechanisms to initiate genome replication, that include de novo initiation, priming by proteins or by self generated 39–ends of templates, and ‘cap–snatching’, among other mechanisms [2]. Protein–primed initiation of genome replication is used by several DNA and RNA viruses and some linear plasmids [3,4,5]. The protein–primer VPg remains bound to the genomic RNA encapsidated into viral particles. Picornaviruses encode only one copy of VPg, except foot–and–mouth disease virus (FMDV) that expresses three similar but non–identical copies of VPg (VPg1–3 or 3B1–3) [8] (Figure 1). FMDV encoding only VPg3 is infectious in cell culture, showing that one copy of VPg may be sufficient to complete the virus replication cycle [12]. Viral replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown
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