mCherry-MNV VPg(102–124) co-immunoprecipitates with translation initiation proteins.

(A) RFP-Trap immunoprecipitation of lysates of BV2 cells expressing mCherry, mCherry-MNV VPg(102–124) WT or mCherry-MNV VPg(102–124) F123A. Pre-purification lysates (Input), purified fractions (IP) and unbound fractions (Flow through) were analysed by SDS PAGE and western blotting. (B) Time course of BV2 infection (MOI 0.01 TCID₅₀ units/cell) with MNV1 in the presence of mCherry-VPg proteins. Prior to infection BV2 cells were transduced with lentiviruses expressing mCherry, mCherry-MNV VPg(102–124) WT or mCherry-MNV VPg(102–124) F123A. The progress of infection was monitored by measuring the number of viral cDNA copies generated from quantitative RT-PCR analysis of whole-cell RNA. (C) Fluorescence microscopy analysis of cell penetration of the FITC-TAT-MNV VPg(102–124) peptides. The images shown are merged images of DAPI stained nuclear DNA (blue) and wild-type or F123A versions of the cell penetrating peptides (green). (D) Time course of BV2 infection (MOI 0.01 TCID₅₀ units/cell) with MNV1 following pre-treatment for 150 minutes with 100 μM of wild-type or F123A versions of the cell penetrating peptides prior to infection. The progress of infection was monitored by RT-PCR analysis (as in panel B).