Table 1.
Prevalence of amino acid residues at position 37 of the M1 protein in H9N2 influenza virus and its reassortants isolated from humans.
Fig 1.
M1 protein with T37A mutation confers increased pathogenicity and replication of H9N2 virus in mice.
Six-week-old female BALB/c mice were individually inoculated with 106 TCID50 of rH9N2:M1-37T or rH9N2:M1-37A viruses, or were mock infected with PBS via the intranasal route. (A) Body weight changes in mice over a 14-day period (n = 5 per group in one independent experiment). Each data point represents the mean ± standard deviation (SD) and is representative of three independent experiments. Statistical significance was based on two-way ANOVA (***P<0.001). (B) Survival rate of mice. Mice that lost more than 30% of their baseline weight were euthanized. Data were pooled from three independent experiments with n = 5 mice per group. Survival curves were compared using log-rank (Mantel–Cox) analysis. **P<0.01. (C) Representative hematoxylin and eosin (H&E)-stained and immunohistochemistry (IHC) examination of lung sections from three mice infected with each indicated virus at 3 days post-infection (dpi). Arrows indicate viral NP localizations. Scale bar, 200 μm. (D) Virus titers recovered from mouse lungs and turbinates, harvested from three mice per group at 3, 5, and 7 dpi. Each data point represents the mean ± SD and is representative of three independent experiments. Dashed lines indicate the lower limit of detection. Statistical significance was based on two-way ANOVA (*P<0.05; **P<0.01; ***P<0.001).
Fig 2.
The T37A substitution in M1 protein is associated with increased production of H9N2 virus in human A549 cells.
A549 cells were infected with the rH9N2:M1-37T or rH9N2:M1-37A virus at an multiplicity of infection (MOI) of 0.2 (A, B) or 1 (C, D). (A, C) Virus output of rH9N2:M1-37T and rH9N2:M1-37A viruses from infected A549 cells. Multistep growth curves of H9N2 viruses (A) and single replication cycle of H9N2 viruses (C). Virus titers were determined from supernatants collected at the indicated time points. Data are presented as the mean ± SD of three independent experiments. Statistical significance was based on two-way ANOVA (*P<0.05; **P<0.01; ***P<0.001). (B, D) Viral M1 and NP production in A549 cells. A549 cells were infected with the rH9N2:M1-37T or rH9N2:M1-37A virus and harvested at the indicated time points; western blotting (WB) was then performed on the cell lysates. The panels show viral protein expression at 12, 24, 36, and 48 hours post-infection (hpi) (B), or at 4, 6, 8, and 10 hpi (D). β-Actin was used as a loading control. The WB results are representative of three independent experiments.
Fig 3.
The stability of H9N2 M1 protein is increased by the replacement of T37 with A37.
(A) The protein abundance levels of M1-37T and M1-37A at different transfection doses. HEK293T cells were co-transfected with the indicated dose of Flag-tagged M1 (37T or 37A) and pEGFPC1 plasmids. Western blotting (WB) was performed to analyze the expression levels of Flag-M1 and GFP. GFP was used as a transfection control and α-Tubulin was used as a loading control. (B–G) Protein degradation assay of M1 protein. (B, C) A549 cells were transfected with Flag-tagged M1 (37T or 37A) expression plasmids for 24 h, followed by CHX (50 μg/mL) treatment over the indicated time course. (D, E) Protein degradation assay of M1 protein in the stable M1-overexpressing A549 cell lines (A549-M1-37T and A549-M1-37A). (F, G) A549 cells were infected with rH9N2:M1-37T or rH9N2:M1-37A for 24 h and then treated with CHX. NP protein was used as a viral protein control, and α-Tubulin was used as a loading control. Densitometry analysis of the data presented in Fig B, D, and F are displayed as respective graphs (Fig C, E, and G), and 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). (H, I) WB analysis to detect the expression levels of M1 in A549 cells transfected with Flag-tagged M1 (37T or 37A) plasmids for 24 h, followed by MG132 (20 μM, H) or CQ (150 μM, I) treatment for 6 h. (J) Ubiquitination analysis of M1. HEK293T cells were co-transfected with Ha-tagged ubiquitin (Ha-Ub) plasmids and Flag-tagged M1 or empty vector plasmid; 24 h later, the cells were treated with MG132 (20 μM) for 6 h. Ubiquitinated proteins were then analyzed by WB. (K) HEK293T cells were co-transfected with Flag-tagged M1 (37T or 37A) or empty vector plasmid, and plasmid encoding a version of ubiquitin capable of binding the substrate only through K63; 24 h later, the cells were treated with MG132 for 6 h. Ubiquitinated proteins were then analyzed by WB. All WB results are representative of three independent experiments.
Fig 4.
T37 of the H9N2 M1 protein is phosphorylated by PKG.
(A) Immunoblotting analysis (IB) analysis of the threonine phosphorylation levels of M1 protein. HEK293T cells transfected with Flag-tagged M1 (37T or 37A) expression plasmids or empty vector (Flag-EV) plasmid for 24 h, followed by MG132 (20 μM) treatment for 6 h, were immunoprecipitated with Flag beads. Threonine phosphorylation levels of M1 were detected by an anti-phosphothreonine antibody. (B) Interaction between M1 and PKG. IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged M1 (37T or 37A) and Ha-tagged PKG expression plasmids. (C) In vitro kinase assay. Bacterially-purified GST-M1 (37T or 37A) and purified Flag-PKG from HEK293T cells were incubated in the presence of ATP and cGMP in vitro. IB was performed to analyze the threonine phosphorylation levels of M1, GST-M1, and Flag-PKG. (D) IB analysis of the threonine phosphorylation levels of M1 in the immunoprecipitates of control or PKG-silenced A549Flag-PKG cells infected with rH9N2:M1-37T or rH9N2:M1-37A for 24 h. α-Tubulin was used as a loading control. The western blotting results are representative of three independent experiments.
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.
Fig 6.
The T37A substitution increases both H5N6 M1 protein stability and H5N6 viral replication in mammals.
(A) The protein expression levels of H5N6 M1 proteins at different transfection doses. HEK293T cells were transfected with the indicated dose of Ha-tagged H5N6 M1 (H5N6-M1-37A or H5N6-M1-37T) expression plasmids. Western blotting (WB) was performed to analyze the expression levels of Ha-M1. α-Tubulin was used as a loading control. (B–E) Protein degradation assay of H5N6 M1 protein. A549 cells were transfected with Ha-tagged H5N6 M1 (37A or 37T) expression plasmids (B) or were infected with rH5N6:M1-37A or rH5N6:M1-37T (D) for 24 h, followed by CHX (50 μg/mL) treatment over indicated time course. α-Tubulin was used as a loading control. Densitometry analysis of the data presented in (B, D) are displayed in respective graphs (C, E), and 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). (F) WB analysis of the expression levels of H5N6-M1 in A549 cells transfected with Ha-tagged H5N6 M1 plasmids followed by MG132 (20 μM) treatment for 6 h. (G) WB analysis of the half-life of H5N6 M1 protein in control or PKG-silenced A549Flag-PKG cells. Control or PKG-silenced A549Flag-PKG cells were transfected with Ha-tagged H5N6-M1-37T plasmid for 24 h, then treated with CHX for the indicated time. WB was performed to analyze the expression levels of Ha-M1 and Flag-PKG. Data were quantified as the ratio of Ha-M1 to α-Tubulin and are displayed in graph (H). 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). All WB data are representative of three independent experiments showing similar results. (I) Virus output of rH5N6:M1-37T and rH5N6:M1-37A viruses from infected A549 cells. A549 cells were infected at a multiplicity of infection (MOI) of 0.001. Virus titers were determined from supernatants collected at the indicated time points. Error bars represent the SD from three independent experiments. Statistical significance was based on two-way ANOVA (*P<0.05). (J, K) Virus titers recovered from mouse lungs (J) and turbinates (K). Lungs and turbinates were harvested from three mice per group at 3, 5, and 7 dpi for virus titration. Each data point represents the mean ± SD and is representative of three independent experiments. Dashed lines indicate the lower limit of detection. Statistical significance was based on two-way ANOVA (*P<0.05; **P<0.01).
Fig 7.
M1 protein harboring T37 is susceptible to ubiquitination at K187.
(A–D) Western blotting (WB) to analyze the half-life of M1-37T and M1-37T/187A protein in A549 cells (A), and the half-life of M1-37T/187A protein in control or PKG-silenced A549Flag-PKG cells (C). Cells were transfected with the indicated Ha-tagged M1 plasmids for 24 h and then treated with CHX for the indicated time. WB was performed to analyze the expression levels of Ha-M1 and Flag-PKG. α-Tubulin was used as a loading control. Densitometry analyses of the data in (A, C) was displayed in respective graphs (B, D), and the data represent the mean ± SD pooled from three independent experiments. Statistical significance was based on two-way ANOVA. The WB data are representative of three independent experiments showing similar results. (E) The effect of PKG on ubiquitination of M1 proteins. HEK293T cells were transfected with Ha-Ub plasmid, GFP-PKG, and indicated Flag-M1 or empty plasmids for 24 h and then treated with MG132 (20 μM) for 6 h. Ubiquitinated proteins were then analyzed by WB. WB results are representative of three independent experiments. (F) Virus output from infected A549 cells. A549 cells were infected with rH9N2:M1-37T, rH9N2:M1-37T/187A, rH9N2:M1-37A, or rH9N2:M1-37A/187A at a multiplicity of infection of 0.2. Virus titers were determined from supernatants collected at the indicated time points. Data are presented as the mean ± SD of three independent experiments. Statistical significance was based on two-way ANOVA (***P<0.001). (G) A549Flag-PKG cells were transfected with control or PKG siRNA for 24 h and then infected by rH9N2:M1-37T, rH9N2:M1-37T/187A, rH9N2:M1-37A, or rH9N2:M1-37A/187A for 24 h. Virus titers in the collected supernatants were analyzed by determining the TCID50. Error bars represent the SD from the mean for three independent experiments. Statistical significance was determined by t-tests (***P<0.001).