Figure 8
From: The C-terminal amyloidogenic peptide contributes to self-assembly of Avibirnavirus viral protease
Protease activity of VP4 with C-terminal MA deletion induces cell death.
(a) Western blotting of in vitro proteolytic assay of polyprotein VP243 synthesized by the TNT system. Plasmids pCI-wtVP243 and pCI-VP243-∆MA were used for in vitro translation to produce polyprotein VP243 and empty plasmid pCI-neo was used as negative control (CK). TNT products were subjected to Western blotting using anti-VP4 and anti-VP3 mAbs. No VP3 or VP4 protein bands were detected in the product of template pCI-VP243-∆MA. (b) Confocal analysis of DF-1 cells transfected with pCI-wtVP243 and pCI-VP243-∆MA. The transfected cells were fixed after 24 h and incubated with a mixture of rabbit anti-VP3 antiserum and mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rabbit IgG. Nuclear DNA was stained with DAPI. (c) Substitution of protease activity site S140 or K180 of VP4 or VP4-C∆2 with alanine (A) failed to induce cell death in transfected DF-1 cells. DF-1 cells transfected with indicated mutants were directly observed under microscopy at 24 h following transfection. Empty plasmid pEGFP-C2 was used as control.
