|Institution:||University of Texas – Austin|
|Keywords:||Influenza B virus; NS1 protein; Effector domain; Brd2 protein|
|Full text PDF:||http://hdl.handle.net/2152/6615|
Influenza B virus is a major human pathogen causing highly contagious respiratory disease. It accounts for approximately ~30% of influenza virus infection per year. The effector domain of the NS1 protein of influenza B virus (NS1B protein), encompassing the carboxy terminal two thirds of the protein, suppresses interferon-β (IFN-β) synthesis in virus-infected cells by unknown mechanism(s). The induced IFN-β mediates innate immunity. To elucidate the mechanism by which the NS1B effector domain suppresses the production of IFN-β, we identified cellular proteins that interact with the NS1B effector domain. Two approaches were used. The approach that succeeded employed the transfection into cells of plasmids expressing the NS1B effector domain containing two affinity tags. After double affinity purification, co-purified cellular proteins were identified by mass spectrometry. We identified Brd2 as a cellular protein that interacts with the NS1B protein. We established that Brd2 specifically binds to the NS1B effector domain in vitro, in vivo, and in virus-infected cells. Serial mutagenesis experiments showed the phenylalanine at position 171 (F171) of the NS1B protein is essential for Brd2 binding. To determine the function of the interaction of Brd2 with the NS1B protein, we generated a recombinant virus encoding an NS1B protein in which F at position 171 was replaced by an alanine. The F171A mutant virus was attenuated, and unlike the wild-type virus, induced the synthesis of IFN-β mRNA. IRF3, a key transcription factor for transcription of the IFN-β gene, was activated in mutant virusinfected cells, but not in wild-type virus-infected cells. Transfection assays implicated the activation of the TBK1 kinase as the step in IRF3 activation that is induced in mutant virus-infected cells. We interpreted these results as showing that Brd2 binding to the NS1B protein is required for suppressing IRF3 activation and IFN-β induction. Attempts at further confirmation by depletion of endogenous Brd2 using RNA interference were not successful because of inefficient knock-down efficiency and nonspecific IFN-β induction. A further complication is that another bromodomain protein, Brd4, interacts with the NS1B protein and could compensate for depletion of Brd2.