Fig. 3.

A small base-paired stretch and the 5′-triphosphate are necessary and sufficient to activate RIG-I. (A) Chemically synthesized 5′-triphosphate 2.2 RNA (syn-ppp-2.2s) either alone or after hybridization (syn-ppp-2.2ds) to 5′-OH-ss2.2as RNA (Left) and chemically synthesized 2.2 hairpin RNA with (syn-ppp-2.2hp) or without (5′-OH-2.2hp) a 5′-triphosphate (Right) was transfected into human monocytes. IFN-α secretion was analyzed after 36 h. (B) Activation of ATP hydrolysis by full-length RIG-I protein was studied with chemically synthesized RNAs bearing either a 5′-OH group or a 5′-ppp group. (C) syn-ppp-2.2s RNA was hybridized to complementary OH-RNA of variable length. The length gradually increased from either the 3′- or the 5′-end. IFN-α production was assessed in human monocytes as in A. (D) 1205Lu human melanoma cells were stimulated with chemically synthesized RNAs after treatment with the indicated siRNAs for 48 h. IP10 levels were measured after 12 h by ELISA. (E) RNA oligonucleotides used in C were examined for their induction of RIG-I ATPase activity in full-length protein. (F) 1205Lu cells stimulated with total RNA isolated from BHK cells infected or not with VSV for 16 h at a multiplicity of infection of 0.2. RNA isolates from VSV-infected BHK cells were treated with equal activities of the RNases III or RNase R before transfection. IP10 levels were determined by ELISA 14 h after stimulation.