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. 2023 Jan 5;14(1):e03231-22. doi: 10.1128/mbio.03231-22

FIG 8.

FIG 8

STRN- and STRN3-mediated support of IAV replication. (A) 293T cells transfected to express STRN or STRN3 or empty vector (EV) as a control. (Upper) One fraction of the cells was infected with SC35M (MOI = 0.001), and viral titers were determined at the indicated time points. Bars indicate means ± SD obtained from three independent experiments performed in triplicates. *, P ≤ 0.05; **, P ≤ 0.01. (Lower) Another fraction of the cells was analyzed by immunoblotting for expression of STRN or STRN3. (B) MLE-15 cells were seeded and transfected 1 and 3 days later with siRNAs targeting mRNAs encoding STRN or STRN3 or an adequate scrambled control as shown. (Left) After 3 days cells, remained untreated or were infected with SC35M (MOI = 0.001) as shown. The cells were infected with SC35M (MOI = 0.001) and viral titers of cell culture supernatants were determined at the indicated time points. Bars indicate means ± SD obtained from three independent experiments performed in triplicates. *, P ≤ 0.05; **, P ≤ 0.01; unpaired t test. (C) The knockdown cells generated in panel B were tested by Western blotting for adequate knockdown and expression of viral proteins as shown; the position of a nonspecific band is highlighted by an asterisk. The lower part shows quantification of three independent experiments; expression in the controls was set to 1, and means ± SEM are indicated. (D) The expression of STRN and STRN3 in 293T cells was reduced by siRNA-mediated knockdown, and cells were infected with SC35M (MOI = 3). (Left) At 6 hpi, cells were treated with DSG and lysates were analyzed for M1 multimerization by Western blotting, similar to Fig. 3C and D. (Right) Three independent experiments were used to calculate the intensity ratios of polymeric to monomeric M1 proteins. Means ± SEM are indicated. (E) Schematic summary of possible functions of M1 T108 phosphorylation investigated in this study. M1 T108 phosphorylation is required for the complete nuclear import of M1 and nuclear export of vRNP, and it affects the ability of M1 for homopolymerization, a process that is also controlled by the STRIPAK complex.