Potyviral VPg Enhances Viral RNA Translation and Inhibits Reporter mRNA Translation In Planta

Abstract

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5 untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

Ribosomal Protein P0 Promotes Potato Virus A Infection and Functions in Viral Translation Together with VPg and eIF(iso)4E

“…We reported previously that VPg requires the PVA 5=-UTR to increase viral translation when expressed in trans but that the PVA 5=-UTR alone is not sufficient to transfer this response to a monocistronic transcript (40), similarly to our results for P0. We therefore analyzed whether P0 and VPg were connected in promoting viral translation.…”

“…The viral untranslated regions (UTRs) appear to promote PVA RNA translation, as the removal of either UTR reduced viral protein accumulation (40). We analyzed the importance of viral UTRs in the P0 promotion of viral translation by expressing exogenous P0 with PVA ⌬GDD lacking either the 5=-UTR…”

“…Luciferase quantitation of virus-derived Renilla luciferase (RLUC) and control FLUC and quantitative RT-PCR (qPCR) were described previously (37,40). Details of Agrobacterium OD 600 s used to transform PVA and other expression cassettes, time points of data collection, and other relevant information are given in the respective figure legends and the text.…”

“…Pprotein-silencing vectors were constructed by generating Gateway-compatible N. benthamiana P-protein cDNA fragments, which were inserted into pHELLSGATE8 (pHG8) (41) via pDONR/Zeo; empty pHG8 was used as a control. The silencing constructs for eIF4E and eIF(iso)4E were described previously (40). All plant expression vectors were used to transform Agrobacterium tumefaciens strain C58C1/pGV2260.…”

“…An eIF(iso)4E Gateway-compatible PCR product was recombined via pDONR/Zeo (Invitrogen) into pGWB18. The GUS and VPg plant expression constructs were described previously (40). Pprotein-silencing vectors were constructed by generating Gateway-compatible N. benthamiana P-protein cDNA fragments, which were inserted into pHELLSGATE8 (pHG8) (41) via pDONR/Zeo; empty pHG8 was used as a control.…”