Fig. 4. m⁶A-deficient hMPVs and virion RNA induce a higher expression of RIG-I.

(a) m⁶A-deficient rhMPVs stimulate a higher expression of RIG-I. A549 cells were infected by each hMPV at an MOI of 0.2, 1.0, and 5.0. At indicated times, cell lysates were subjected to Western blot analyses using antibody specific to RIG-I, hMPV N, or β-actin. (b) m⁶A-deficient virion RNA induces a higher expression of RIG-I. A549 cells were transfected with an increasing amount of poly (I:C) (0.5 and 2.0 µg/well) or virion RNAs (2×10⁵, 2×10⁶, or 2×10⁷ copies/well) of rhMPV, rhMPV-G8-14, or rhMPV-G1-14. At indicated times, cell lysates were subjected to Western blot analysis using antibody against RIG-I or β-actin. (c) Comparison of RIG-I expression triggered by virion RNA. A549 cells were transfected with increasing amounts (10⁵, 10⁶, and 10⁷ RNA copies) of virion RNA of rhMPV, rhMPV-G1-2, rhMPV-G8-9, or rhMPV-G1-14. (d) Removal of 5’ triphosphate abolished RIG-I expression and IRF3 phosphorylation. A549 cells were transfected with virion RNA with or without CIP treatment. At indicated times, cell lysates were subjected to Western blot using antibody specific to IRF3 or phosphorylated IRF3 (pIRF3) on site S386 or S396. (e) Natural m⁶A-deficient rhMPVs induce higher phosphorylation of IRF3. A549 cells were infected by each hMPV at an MOI of 5.0. At indicated times, RIG-I expression and IRF3 phosphorylation was detected by Western blot. (f) Natural m⁶A-deficient virion RNA induces higher RIG-I expression. 10⁷ copies of virion RNA were used for transfection. Western blots (a-f) shown are the representatives of n =3 biologically independent experiments. (g) Model for RIG-I mediated IFN signaling pathway. Upon hMPV entry, the RNP complex is delivered into the cytoplasm where RNA synthesis and viral replication occur. During replication, the RdRP initiates at the extreme 3’ end of the genome and synthesizes a full-length complementary antigenome, which subsequently serves as template for synthesis of full-length progeny genomes. The newly synthesized genome and antigenome was methylated by m⁶A writer proteins and encapsidated by viral N protein. Viral genome and antigenome are recognized by cytoplasmic RNA sensor RIG-I and induces signaling to the downstream adaptor protein MAVS which subsequently activates IRF3 and NF-κB pathways, leading to the production of type-I IFN. The internal m⁶A methylation on virion RNA inhibits RIG-I mediated IFN signaling pathway.