Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion
Fig 5
Molecular interaction between M and methyltransferase domain of METTL3.
(A) 293T cells were cotransfected with M expression plasmid together with FLAG-METTL3 expression plasmid. For infected cells, the cells were transfected with FLAG-METTL3 expression plasmid and infected with rBPIV3-EGFP at an MOI of 1 at 24 h post-transfection (hpt). At 72 hpt, the cells were harvested and subjected to coimmunoprecipitation with anti-FLAG antibody (Ab). The precipitates were analyzed by western blotting using anti-BPIV3-M Ab. (B) Schematic representation of human METTL3. ZF1 and ZF2: two zinc finger domains, MTD: methyltransferase domain, DPPW: DPPW motif. The 1-400, 1-380, 1-200, and 161-580 deletion mutants lacking each domain are shown. 293T cells were cotransfected with the deletion mutants along with BPIV3-M. At 48 hpt, the cells were harvested and subjected to coimmunoprecipitation with anti-FLAG Ab as described in the legend of Fig 5A. (C) Schematic representation of FLAG- and GST-tagged deletion mutants of the BPIV3 M protein used to map the METTL3 interaction domain. Cells were transfected with a FLAG-METTL3 expression plasmid, and cells were harvested at 48–72 h post-transfection. Deletion mutants of the M protein fused with FLAG and GST tags were synthesized using a wheat germ cell-free expression system. Each FLAG-GST-tagged M deletion mutant was incubated with glutathione–sepharose beads at 4°C, followed by the addition of lysates containing FLAG-METTL3. After incubation, the beads were extensively washed, and bound proteins were analyzed by western blotting. Both METTL3 and M deletion mutants were detected using an anti-FLAG antibody. Each experiment was performed at least twice.