Abstract
Vaccinia virus, a large DNA virus that replicates in the cytoplasm, expresses its E3L protein to inhibit the cellular innate immune response and apoptosis. E3L is a bifunctional protein that contains an N-terminal DNA binding domain (BD) and a C-terminal double-stranded RNA (dsRNA)-BD (residues 100-190), both of which contribute to viral pathogenesis by blocking the activation of cellular genes that respond to the viral infection. We report that expression of the dsRNA-BD alone inhibits not only the dsRNA-induced activation of interferon beta (IFNbeta) but also that of 5'-triphosphate single-stranded RNA and DNA-induced IFNbeta activation even though E3L(100-190) does not bind the latter two pathogen-associated molecular patterns. This inhibition occurs in both human HeLa and A549 cells, where RIG-I appears to be required for dsDNA-induced IFNbeta activation. Unexpectedly, the two residues most important for dsRNA binding are also critical for this domain's ability to inhibit all three nucleic acid-induced cellular responses.
Highlights
Lar RNAs are made in the nucleus, and it is thought that essentially only ssRNAs without free 5Ј-triphosphate ends are allowed to enter the cytoplasm
Vaccinia virus (VV), a DNA virus that replicates in the cytoplasm, encodes the E3L protein that antagonizes the effects of IFNs intracellularly [16]
This paper reports that the vaccinia virus (VV) E3L double-stranded RNA (dsRNA)-binding domain (BD) alone (E3L100–190) inhibits the 5Ј-pppssRNA- and dsDNA-induced activation of IFN in both murine and human cells even though it does not bind to these nucleic acids
Summary
Viruses, and Antibodies—HeLa, A549, and MEF cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. RSeV-GFP(ϩ), which expresses green fluorescent protein (GFP) from a transgene between the M and F genes, and rSeVE3L100 –190, which expresses E3L100 –190 from a located transgene, were prepared as previously described [23]. P-IFN-fl-lucter, which contains the firefly luciferase gene under the control of the human IFN- promoter, was described previously [25]. PTK-rl-lucter, used as a transfection standard, contains the herpes simplex virus TK promoter region upstream of the Renilla luciferase gene (Promega). Transfections—100,000 cells were plated into 6-well plates 20 h before transfection with 1.5 g of p-IFN-fl-lucter, 0.5 g of pTK-rl-lucter, 1 g of plasmids expressing RIG-I, E3L (according to the various figure legends), and TransIT-LT1 transfection reagent (Mirus). At 24 h post-transfection, the cells were (or were not) infected with various SeV stocks or transfected with 5 g of poly(I-C) (sigma) or 10 g of poly(dAdT) using TransIT-LT1 transfection reagent.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
