Enhanced stability of M1 protein mediated by a phospho-resistant mutation promotes the replication of prevailing avian influenza virus in mammals
Fig 5
Loss of T37 phosphorylation protects M1 protein from degradation directed by PKG.
(A) HEK293T cells were co-transfected with Flag-tagged M1 (37T or 37A), pEGFPC1, and an increasing dose of Ha-PKG expression plasmids. Western blotting (WB) was performed to analyze the expression levels of M1, GFP, and PKG. (B) WB analysis of the expression levels of Ha-M1, Flag-PKG, and GFP in control or PKG-silenced A549Flag-PKG cells transfected with Ha-tagged M1 and GFP expression plasmids. GFP was used as a transfection control and α-Tubulin was used as a loading control. (C–F) WB analysis of the half-life of M1 protein in control or PKG-silenced A549Flag-PKG cells transfected with M1 expression plasmids. Control or PKG-silenced A549Flag-PKG cells were transfected with Ha-tagged M1-37T (C) or M1-37A (E) expression plasmids for 24 h, then treated with CHX (50 μg/mL) for the indicated time. WB was performed to analyze the expression levels of Ha-M1 and Flag-PKG. Data presented in Fig C and E were quantified as the ratio of Ha-M1 to α-Tubulin and were displayed in respective graphs (D, F). The data represent the mean ± SD pooled from three independent experiments. Statistical significance was based on two-way ANOVA (**P<0.01; ***P<0.001). (G) The effect of PKG on the ubiquitination of M1 proteins. Ha-Ub plasmids were co-transfected in HEK293T cells with GFP-PKG and Flag-M1 or empty plasmids for 24 h, followed by MG132 (20 μM) treatment for 6 h. Ubiquitinated proteins were then analyzed by WB. (H) WB analysis of M1 and PKG expression in A549 cells that had been transfected with Flag-PKG expression plasmids for 24 h, followed by rH9N2:M1-37T or rH9N2:M1-37A virus infection for 24 h. (I) Virus titers in supernatants, as described in (H), were analyzed to determine the TCID50. (J) WB analysis of M1 and Flag-PKG in A549Flag-PKG cells transfected with control or PKG siRNAs for 24 h, followed by rH9N2:M1-37T or rH9N2:M1-37A infection for 24 h. (K) Virus titers in supernatants, as described in (J), were analyzed by TCID50. Error bars in (I) and (K) represent the SD from the mean for three independent experiments. Statistical significance was based on t-tests (***P<0.001). All WB data are representative of three independent experiments showing similar results.