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. 2012 Nov 29;8(11):e1003059. doi: 10.1371/journal.ppat.1003059

Figure 1. Influenza A Virus NS1 interacts with TRIM25 in a species-specific manner.

Figure 1

(A) HEK293T cells were transfected with V5-tagged mouse TRIM25 (V5-mTRIM25), human TRIM25 (V5-hTRIM25) or chicken TRIM25 (V5-chTRIM25) together with NS1-A/California/04/09 (Cal04) (left), or NS1-PR8, NS1-A/Hong Kong/156 (HK156), NS1-A/Swine Texas/98 (SwTx98), or NS1-Cal04 (right panel). Empty vector and V5-tagged human TRIM21 were used as negative controls. Whole-cell lysates (WCLs) of HEK293T cells were subjected to immunoprecipitation (IP) using anti-V5 antibody, followed by immunoblotting (IB) with anti-NS1. (B–E) TRIM25 is important for virus-induced IFN-β production in chicken cells. Chicken LMH cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for chicken TRIM25 (si.chTRIM25). At 40 h posttransfection, cells were infected with recombinant influenza viruses expressing the NS1 RNA-binding mutant R38A/K41A (C), or NS1 from PR8, SwTx98, or HK156 (D) at an MOI of 0.1 for 24 h. Knockdown was confirmed by determining chTRIM25 mRNA levels (B). Furthermore, the IFN-β (C and D) and viral M1 (E) mRNA levels were assessed by qPCR and the values were normalized to chicken GAPDH. (F) IFN induction and viral replication in murine (MEF) cells. 12 hours postinfection, IFN-β (far left panel) and viral M1 (right panel) mRNA levels were assessed by qPCR from MEFs infected with the different NS1 recombinant viruses at an MOI of 2. Values were normalized to mouse actin. IFN protein was quantified by VSV-GFP bioassay on L929 cells treated with 2-fold dilutions of post-influenza virus supernatants from MEF cells (left panel). Relative fluorescence values reported on the y-axis represent the levels of VSV-GFP replication. High IFN concentrations in the supernatants correspond to low levels of VSV-GFP replication (low fluorescence values). Each sample was assayed in triplicate, and results are representative of two independent experiments. Supernatants were assayed for progeny virus yields 12 h postinfection in standard plaque titrations (far right panel). Virus yields are depicted in PFU/ml. *p<0.05; ***p<0.001.