A Novel Interaction Network Used by Potyviruses in Virus-Host Interactions at the Protein Level.

Host proteins that are central to infection of potyviruses (genus Potyvirus; family Potyviridae) include the eukaryotic translation initiation factors eIF4E and eIF(iso)4E. The potyviral genome-linked protein (VPg) and the helper component proteinase (HCpro) interact with each other and with eIF4E and eIF(iso)4E and proteins are involved in the same functions during viral infection. VPg interacts with eIF4E/eIF(iso)4E via the 7-methylguanosine cap-binding region, whereas HCpro interacts with eIF4E/eIF(iso)4E via the 4E-binding motif YXXXXLΦ, similar to the motif in eIF4G. In this study, HCpro and VPg were found to interact in the nucleus, nucleolus, and cytoplasm in cells infected with the potyvirus potato virus A (PVA). In the cytoplasm, interactions between HCpro and VPg occurred in punctate bodies not associated with viral replication vesicles. In addition to HCpro, the 4E-binding motif was recognized in VPg of PVA. Mutations in the 4E-binding motif of VPg from PVA weakened interactions with eIF4E and heavily reduced PVA virulence. Furthermore, mutations in the 4G-binding domain of eIF4E reduced interactions with VPg and abolished interactions with HCpro. Thus, HCpro and VPg can both interact with eIF4E using the 4E-binding motif. Our results suggest a novel interaction network used by potyviruses to interact with host plants via translation initiation factors.

Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells.

Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae) encodes a Class 1 RNase III endoribonuclease (RNase3) that suppresses post-transcriptional RNA interference (RNAi) and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas). For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA) and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6). In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression.

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif.

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.

VPg of Potato virus A alone does not suppress RNA silencing but affects virulence of a heterologous virus.

The viral genome-linked protein (VPg) is a well-known virulence factor in potyviruses (genus Potyvirus), including Potato virus A (PVA). Its ability to suppress onset and signalling of transgene-mediated RNA silencing and accumulation of small interfering RNA (siRNA) was studied using cross-protection and Agrobacterium infiltration assays and green fluorescent protein (GFP) and PVA VPg protein-expressing transgenic Nicotiana benthamiana plants. N. benthamiana plants were also transformed with a transgene comprising the cylindrical inclusion protein (CI), nuclear inclusion protein a (NIa) and coat protein (CP) encoding regions of PVA. This transgene mRNA was expressed in the T1 progeny of the transgenic lines but all were susceptible to PVA. This result contrasted the plants transformed with the PVA P1, VPg (N-proximal part of NIa) or CP encoding regions that expressed various forms of resistance. There was little evidence for direct involvement of VPg in suppression of silencing, while other mechanisms by which VPg might interfere with transgenic resistance could not be excluded. Expression of the wild-type PVA VPg from the genome of Potato virus X (PVX, genus Potexvirus) increased symptom severity in N. benthamiana, whereas a single point mutation introduced to the VPg enhanced accumulation of the PVX chimera. These data demonstrated previously unknown virulence functions controlled by the VPg of a potyvirus.