Fig. 1.

PDZ Motifs 29-DEMI-32 and 39-KEALSDGI-46 are important for hMPV-induced innate immune responses. (A) Immune mediator induction by rhMPV. A549 cells in triplicate were mock infected or infected with rhMPV-WT, rhMPV-E30M31, or rhMPV-E40L42D44, at MOI of 2, for various time as indicated. The secretion of cytokines and chemokines in cell supernatants were measured by Bio-plex and/or ELISA. Data shown are from three independent experiments and are expressed as mean± SE. *, P<0.05, and **, P<0.01, relative to rhMPV-WT-infected A549 cells. (B) NF-κB and IRF-3 activation by rhMPV. A549 cells in flasks were mock infected or infected with rhMPV as described in A. nuclear fractions were prepared, followed by western blot to assess the nuclear translocation of p65 and IRF-3. The expression of a nuclear protein lamin B, used as an internal control, was also compared among the group. (C) Replication and gene transcription characterization of recombinant viruses. A549 cells in 6-well plates were mock infected or infected with rhMPV at MOI of 2 for various periods of time as indicated, followed by total RNA extraction using Trizol. The extracted RNAs in triplicate were then subjected to real-time PCR to assay genomic RNAs (left panel) or viral G gene transcription (right panel). The results are the representative of two independent experiments and are expressed as mean±SE of absolute copy numbers of transcribed G gene or viral genome. **,P<0.01, relative to rhMPV-WT-infected A549 cells. (D) Impact of motifs 29-DEMI-32 and 39-KEALSDGI-46 on M2-2-mediated immune evasion. A549 cells in triplicate were transfected with a luciferase reporter plasmid IFN-β-Luc (0.1 µg/well), a plasmid encoding MAVS or its control (0.1 µg/well), and a plasmid encoding hMPV M2-2 or its mutants, or a control vector. (0.1 µg/well) were transfected. After 40 h, cells were harvested for luciferase activity measurement. *P<0.05 and P<0.01 relative to MAVS+M2-2. CV: control vector for M2-2 or MAVS expression.