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. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/6615 |
| Date | 22 October 2009 |
| Creators | Park, Jang Won |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Format | electronic |
| Rights | Copyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works. |
Page generated in 0.0019 seconds