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. Author manuscript; available in PMC: 2018 Dec 28.
Published in final edited form as: Cell Host Microbe. 2017 Nov 5;22(5):627–638.e7. doi: 10.1016/j.chom.2017.10.003

Figure 1. TRIM25 inhibits influenza viral replication in infected cells.

Figure 1.

(A) CRFK cells engineered to stably express the indicated FLAG-tagged TRIM25 proteins, or FLAG-TRIM5α (human) as a negative control. Immunoblots were probed with mouse anti-FLAG antibody and mouse anti-βActin antibody. (B) The CRFK cells from panel A were infected with 0.2 pfu/cell of influenza A Udorn virus. Whole cell extracts collected at 8 hours post infection were analyzed by immunoblotting with an anti-Udorn goat polyclonal antibody recognizing HA, NP, and M1 (Chen et al., 2007). Quantitation of immunoblots was performed using ImageJ software, and the percent reduction in viral protein levels relative to the vector control are shown. The values for percent reduction correspond to the average change in the levels of HA, NP and M1. Each viral protein band was normalized using the actin control. (C) CRFK cell lines expressing human TRIM25 were pre-treated with the indicated amounts of wildtype (wt) NS1-expressing retrovirus, 38/41 mutant NS1-expressing retrovirus, or a control retrovirus (ctl). These cells were then infected with 0.2 pfu/cell of Udorn virus. A cell extract collected from mock infected cells was immunoblotted using rabbit anti-NS1 antibody as a reference for the amount of NS1 present at the time of infection. Extracts of cells collected at 8 hours post infection were immunoblotted using the anti-Udorn polyclonal antibody. Quantitation of immunoblots was performed as in panel B, with fold rescue relative to the sample receiving control retrovirus reported. Data presented in panels B and C are representative of two experimental replicates. (D) Virus replication after infection of the indicated CRFK cells with 0.2 pfu/cell of Udorn virus. Error bars represent standard error from three independent experiments. Asterisks indicate significant one-way ANOVA comparisons (*p < 0.01). For each time point, a post-hoc Tukey-Kramer test found all pairwise comparisons significant with p < 0.05. (E) qRT-PCR measurements of HA, NP, and NS1 mRNA and vRNA levels at 7 hours after infection of the indicated CRFK cell line with 0.2 pfu/cell of Udorn virus. Expression is normalized to GAPDH mRNA level. Error bars represent standard deviation of three independent experiments. Each mRNA/vRNA group passed a one-way ANOVA test with p < 0.01. Asterisks indicate significant (*p < 0.01) pairwise comparisons in a post-hoc Tukey-Kramer test. See also Figures S1-S3.