A Functional Link between RNA Replication and Virion Assembly in the Potyvirus Plum Pox Virus

Abstract

Accurate assembly of viral particles in the potyvirus (PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced in leaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro supplied , was assembled into virus-like particles and remained stable after incubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential coagroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads in leaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a nonreplicating viral RNA can be stably assembled into virions as long as it is coexpressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection. Viruses of the family are among the most dangerous threats for basically every important crop, and such socioeconomical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case of (genus ), for instance, we have previously shown that the multitasking viral factor HCPro plays a role in the production of stable virions. Here, by using this potyvirus as a model, we move further to show that additional factors are also necessary for the efficient production of potyviral particles. More importantly, a comprehensive screening for such factors led us to the identification of a functional link between virus replication and packaging, unraveling a previously unknown connection of these two key events of the potyviral infection cycle.

Insights into the Functions of eIF4E-Binding Motif of VPg in Potato Virus A Infection

“…We Agrobacterium-infiltrated Nicotiana benthamiana plants with PVA VPgmut and PVA WT , and we collected leaf samples from the infiltrated leaves at 7 and 10 dpi. In the light of the current knowledge, the formation of virus particles is a strong proof for potyvirus replication [53]. However, in contrast to PVA WT , we could not detect any particles from the local leaves of PVA VPgmut -expressing plants by electron microscopy at 7 dpi ( Figure 3A).…”

“…PVA RNA that is encapsidated to PVA particles carries VPg at its 5'end [51]. Potyviral RNA is encapsidated only if it is able to replicate [52,53]. If PVA VPgmut replicates, according to the same logic, PVA VPgmut RNA ought to carry VPg mut protein at its 5'end, as schematically drawn in Figure 2A.…”

Plant Virus Genome Is Shaped by Specific Dinucleotide Restrictions That Influence Viral Infection

“…Plants were grown in a greenhouse with 16-h-light/8-h-dark cycles at 20 to 24°C for N. benthamiana and P. persica GF305 and 8-h-light/16-h-dark cycles at 21°C for A. thaliana. Leaves of 4-week-old plants were infiltrated using Agrobacterium tumefaciens C58C1 strains carrying the indicated plasmids, as previously described (33). Just before agroinoculation, cultures were adjusted to an optical density at 600 nm (OD 600 ) of 0.05 for infiltrations in N. benthamiana, an OD 600 of 0.5 for infiltrations in P. persica, and an OD 600 of 1.0 for infiltrations in A. thaliana.…”

Common and Strain-Specific Post-Translational Modifications of the Potyvirus Plum pox virus Coat Protein in Different Hosts

“…For agroinoculation, young P. persica cv. GF305 and N. benthamiana plants (four-to-six-leaf stage) were infiltrated with cultures of Agrobacterium tumefaciens GV3101 (pMP90, pJIC SA_Rep) transformed with plasmid pSN-PPV-5 BD-GFP, as previously described [50]. In the case of Prunus plants, the agrobacterium pellet was suspended in inoculation buffer to reach an OD 600 of 1, and leaves were infiltrated by pressing strongly and repeatedly on the syringe plunger, in overlapping patches, to cover most of the foliar area.…”