Figure 5. E3 sequesters Z-RNA that accumulates during VACV infection.

(A) Confocal immunofluorescent micrographs of single L929 cells at 4 hpi. Cells were either uninfected (mock) or infected with the indicated viruses at an MOI of 5, fixed and permeabilized prior to staining with anti-Z-NA antibody (green), anti-E3 antibody (red) and DAPI nuclear dye (blue). Bar indicates 10 µM.

(B) Quantification of the median florescence intensity (MFI) of Z-NA-specific monoclonal antibody staining in individual L929 cells using Leica LAS X software. Each point represents an individual cell and mean represents the average intensity from 20 cells.

(C) Confocal immunofluorescent micrographs of single L929 cells at 4 hpi. Cells were either uninfected (mock) or infected with the indicated viruses at an MOI of 5 and fixed and permeabilized then subsequently left untreated or digested with DNase I or RNase A. Cells were stained as described in (A).

(D) Coimmunoprecipitation (coIP) of RNA bound to E3 or ZBP1. SVEC-derived WT ZBP1 cells were left uninfected (mock) or infected with indicated viruses at an MOI of 5. At 5-hpi cells were UV crosslinked, lysed and either ZBP1- or E3-associated RNA was isolated by coimmunoprecipitation with E3-specific antibody (triangles) or FLAG-specific antibody. RNA associates with either of these immunoprecipitates were isolated by Trizol, quantified by nanodrop, and the proportion recovered was compared with total cytoplasmic RNA.

(E) Coimmunoprecipitation of E3 and ZBP1. SVEC-derived WT ZBP1 cells were either left uninfected (mock) or infected at an MOI of 5 with WT VACV (expresses p20 and p25) or E3Δ37N (expresses only p20). Cells were UV crosslinked at 5 hpi, lysed, and cell lysates were subjected to immunoprecipitation with either mouse anti-E3-specific antibody (BEI) or rabbit anti-FLAG antibody (cell signaling) specific for ZBP1 here). Immunoprecipitates were treated with DNase I alone or in combination with RNase T1 and subsequently washed to remove unbound proteins. Total lysates (right panel) and immunoprecipitates were subjected to SDS-PAGE and evaluated for ZBP1 with a mouse anti-ZBP1 antibody (Adipogen) or for E3 with rabbit polyclonal anti-E3 antibody. * indicates specific bands.

(F) CoIP of E3 with anti-Z-NA antibody. SVEC EV cells were either left uninfected (mock) or infected at an MOI of 5 with WT VACV or E3Δ37N. Cells were UV crosslinked at 6 hpi and treated with DNase I. Z-NA was immunoprecipitated from whole cell lysates and total lysates, and immunoprecipitated samples were subjected to SDS-PAGE and IB evaluation with rabbit polyclonal anti-E3 antibody.

Error bars represent the SD. Each set of data is representative of three replicates. Statistical significance was determined as described in Figure 1