VZV ORF20 contains a RHIM that inhibits TNF-induced necroptosis in HT-29 adenocarcinoma cells.

(A) Schematic diagram of RHIM-containing proteins. RHIM location indicated by yellow box. (B) Amino acid sequence alignment of the new RHIM identified in VZV ORF20 (Dumas strain) with other known human cellular RHIMs from RIPK3, RIPK1, TRIF and ZBP1 and the viral RHIMs from MCMV (M45), HSV-1 (ICP-6) and HSV-2 (ICP10), also indicating the percentage of conservation and consensus sequence, with the RHIM core tetrad boxed. (C) Cytopathic effect and immunofluorescence staining for VZV antigens (IE62, pORF29 and the gE:gI glycoprotein complex, green) in VZV-infected and mock HT-29s. Following immunostaining cells were counterstained with DAPI (blue). Scale bars indicate 20 μm (D) Viability of mock and VZV infected HT-29s (72 h post-infection) following treatments with TNF (T; 30 ng/ml), BV-6 (S; 1 μM), z-VAD-fmk (V; 25 μM) and necrostatin-1 (Nec1; 30 μM) alone or in combination as indicated. Data was normalised to DMSO only control. Error bars show standard error of the mean, from 4 independent replicates, statistical significance was determined using a two-way ANOVA.