NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL.
The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.
Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638, Japan.
Masaya
M
Miyazaki
Hiroshi
H
Nishihara
Hideki
H
Hasegawa
Masato
M
Tashiro
Lei
L
Wang
Taichi
T
Kimura
Mishie
M
Tanino
Masumi
M
Tsuda
Shinya
S
Tanaka
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000472:pathogenicity*; D053124:Influenza A Virus, H5N1 Subtype / Q000472:pathogenicity*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017361:Viral Nonstructural Proteins; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; C085820:INS1 protein, influenza virus; D009687:Nuclear Proteins; D017361:Viral Nonstructural Proteins; D048049:Extracellular Signal-Regulated MAP Kinases
3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Cell proliferation; CrkII; CrkL; ERK; GST; HEK; MTT; NS1; NS1-BP; NS1-binding protein; SH; Src-homology; extracellular signal-regulated kinase; glutathione S-transferase; human embryonic kidney; non-structural protein 1