Mechanism Of Viral Genome-linked Protein Affecting The Pathogenesis Of Tobacco Vein Banding Mosaic Virus

The viral genome-linked protein (VPg) of potyviruses is a multifunctional proteininvolved in many process of potyviral life cycles, including replication, cell-to-cell and longdistance movement. VPg is attached to the5′end of the genome and is linked covalently toone end of filamentous virus particle. It interacts and forms complexes with several viral andhost proteins, which regulates the pathogenesis of potyviruses. However, the specificmechanism of VPg affecting the pathogenesis of potyviruses remains largely unknown.Tobacco vein banding mosaic virus (TVBMV) is one species of the largest plant virusgenus Potyvirus. The losses caused by TVBMV have been increasing during the recent years.In this study, we constructed29mutants from TVBMV cDNA clone carrying greenfluorescent protein gene by site-directed mutagenesis and analyzed the effects of thosemutations on viral pathogenicity via agroinfiltration. Interaction with eIF4E is analysed withyeast two-hybrid system (YTHS) and bimolecular fluorescence complementation (BiFC). Themain results are as follows:The pathogenicity of mutants at position Lys42, Lys45and Ser178(K42A, K42E, K42Q,K45A, K45E, K45Q, S178A, S178E, S178K) is consistent with that of wild type TVBMV onplants of Nicotiana benthamiana, but K42A and K45E only induced slight symptom on plantsof N. tabacum, suggesting that the mutation at Lys42and Lys45has host-specificity;Mutation at position Gly54resulted in attenuated virulence on plants of both N. benthamianaand N. tabacum.Mutations at position Leu10, Tyr40and Tyr64(L10A, L10D, L10R, Y40A, Y40D,Y40R, Y64A, Y64D, Y64R) made the virus lose the ability of long distance movement.Among these mutants, Y40D and Y40R lose the ability of intercellular movement. Mutationat position Tyr64affected the replication in inoculated leaves. When it was substituted byphenylalanine (F) and tryptophan (W), the virus could not replicate normally, indicating thatthe Tyr64is indispensable in the process of TVBMV replication.The different mutants at position Leu183and YXXXXL motif had different effects:substitution of Leu183to alanine (L183A) or lysine (L183K) showed more serious symptom than wild type. Conversely, substitution of Leu124to alanine (L124A) showed attenuatedsymptom. In addition, mutation of Leu183to glutamic acid (L183E), mutation of Tyr119toalanine (Y119A), mutation at both Tyr119and Leu124to alanine (Y119A-L124A) could notreplicate in cells of N. benthamiana.TVBMV VPg can interact with itself. However, the VPg mutants losing the ability oflong distance movement can only interact with themselves weakly. TVBMV VPg couldinteract with eIF(iso)4E-2specifically in yeast and plant, while could not interact with eIF4Eand eIF(iso)4E-1. Mutants which lose the capacity of replication also lose the ability ofinteracting with eIF(iso)4E-2, which indicated the interaction between VPg and eIF(iso)4E-2is an essential step in TVBMV replication.The results described above reveal that VPg plays an important role in the infection andpathogenicity of TVBMV, and the interaction between VPg and eIF(iso)4E-2is critical forTVBMV infection. This study furthers our understanding on the mechanism of viralgenome-linked protein affecting the pathogenesis of TVBMV and provides materials andtheoretical guidance for the control of TVBMV and relating viruses.