NS1-BP promotes splicing of influenza A virus M1 mRNA segment to yield proper levels of M2 mRNA and protein.

Abstract

<p>(A) A549 cells were transfected with non-targeting or NS1-BP siRNAs for 48 h and infected with A/WSN/33 at MOI 2 for the indicated time points. Cells lysates were subjected to western blot analysis using antibodies against influenza virus proteins. α-tubulin was used as loading control. Each protein band was quantified by ImageJ and normalized to α-tubulin levels. (B) M1 mRNA and its alternatively spliced products are depicted and the arrowheads show primer positions for detection of various mRNAs in the following experiments. (C, D, and E) Cells were transfected and infected as in A and total RNA was isolated and analyzed by real-time RT-PCR with primers specific to M1, M2, mRNA₃, and M4 mRNAs. Ratios of M2 mRNA to M1 (C) or to mRNA₃ (D), or to M4 (E) over time are presented. (F and G) RNA samples from C were analyzed with primers specific to NS2 and NS1 viral mRNAs. (G) Ratios of NS2 mRNA to NS1 mRNA over time are shown. (H) NS1-BP mRNA levels were measured by real time RT-PCR in samples from C. Error Bars represent mean ± SD (n = 3).</p