Fig 5. Viral protein VPg together with HCpro and other PG components regulates viral translation.

P0^YFP and PVA^CPmut were expressed to induce PGs in N. benthamiana leaves, and the effect of co-expressed RFP (A) or VPg^RFP (B) on P0^YFP-labeled PGs was examined by confocal microscopy three days later. RFP fluorescence was used to verify VPg and control expressions. Scale bar; 100 μm. (C—D) Frequency of cells/mm² containing P0^YFP-labeled PGs in leaf tissues during expression of either RFP or VPg^RFP. PVA^CPmut served as an inducer of the PGs in (C) and HCpro in (D). (E—F) PVA^ΔGDD RLUC activities and RNA levels were determined during co-expression of the vRNA with GUS, HCpro, VPg, VPg and HCpro in N. benthamiana leaves at 3 DAI. (G) PVA^ΔHCΔGDD RLUC activity levels were determined during co-expression of vRNA with GUS, VPg, VPg and either HCpro, 4Ebd-HCpro or sd-HCpro in N. benthamiana leaves at 3 DAI. (H) The capacity of VPg to elevate PVA^ΔGDD translation was analyzed during RNA hairpin (hp)-induced silencing of UBP1 and VCS in N. benthamiana leaves. Empty non-recombined silencing vector (hp-) and phytoene desaturase (PDS) hp-constructs were used as controls. VPg or GUS was expressed with PVA^ΔGDD in the different silencing backgrounds and RLUC activity was determined as a reporter of viral protein expression at 5 DAI. (I) The capacity of UBP1 and UBP1^rrm to affect VPg promoted PVA^ΔGDD RLUC translation was analyzed. Here, GUS or VPg were co-expressed with PVA^ΔGDD during ectopic expression of GUS, UBP1, or UBP1^rrm mutant lacking RNA-binding domains. All quantitative data is presented as mean and the error bar indicates the standard deviation. (J) The effect of UBP1 and VCS silencing and the effect of UBP1^rrm overexpression on PVA-induced PG formation was analyzed by determining the frequency of cells/mm² showing PGs in N. benthamiana leaves at 3 DAI. (p < 0.01 **, p < 0.05 *).