Poly da dt po833

Poly(dA:dT) (PO833) and ribavirin were purchased from Sigma-Aldrich and used at 500 ng/ml and 400 μM final concentration, respectively. Cyclic diguanosine monophosphate (cyclic-di-GMP) was purchased from BioLog Life Science Institute (Bremen, Germany) and used at a concentration of 1 μg/ml. Recombinant IFN-A/D was purchased from PBL Assay Science (Piscataway, NJ).
For production of recombinant RIG-I used in Fig. 2, the 3xFLAG-human RIG-I DNA sequence¹³ was amplified using forward primer 5′-actcgagttatggactacaaagaccatgacgg-3′ and reverse primer 5′-ttgcggccgctcatttggacatttctgctggatcaa-3′ and cloned into the pBacPAK-His3-GST plasmid. Recombinant 3xFLAG-human RIG-I was expressed as a GST-tagged protein in SF9 insect cells using a baculovirus expression system and purified in a single step by affinity chromatography using glutathione-sepharose matrix (GE Healthcare, Little Chalfont, United Kingdom). The protein was eluted by GST tag cleavage using 3C enzymatic digestion. A final polishing step was accomplished using a Superdex 200 10/300 GL column (GE Healthcare). Protein purity was verified by acrylamide gel electrophoresis, and protein yield was quantified using a Nanodrop apparatus (ThermoScientific, Waltham, MA).