Interaction of influenza virus NS1 protein with growth arrest-specific protein 8

Abstract

NS1 protein is the only non-structural protein encoded by the influenza A virus, and it contributes significantly to disease pathogenesis by modulating many virus and host cell processes. A two-hybrid screen for proteins that interact with NS1 from influenza A yielded growth arrest-specific protein 8. Gas8 associated with NS1 in vitro and in vivo. Deletion analysis revealed that the N-terminal 260 amino acids of Gas8 were able to interact with NS1, and neither the RNA-binding domain nor the effector domain of NS1 was sufficient for the NS1 interaction. We also found that actin, myosin, and drebrin interact with Gas8. NS1 and beta-actin proteins could be co-immunoprecipitated from extracts of transfected cells. Furthermore, actin and Gas8 co-localized at the plasma membrane. These results are discussed in relation to the possible functions of Gas8 protein and their relevance in influenza virus release.

Figure 1

In vitro and in vivo interaction between NS1 and Gas8. A GST pull-down analysis. Lysates from cells expressing myc-Gas8 were mixed with GST or GST-NS1, and bound proteins were analyzed by anti-myc western blot. Left lane, cell lysate; middle and right lanes, proteins bound to GST-NS1 and GST, respectively. Gas8 was precipitated by a GST-NS1 fusion protein but not by GST. (B) Western blot of NS1 and Gas8 co-IPs. Left: Cells were co-transfected with a plasmid expressing myc-tagged NS1 and either a plasmid expressing FLAG-tagged Gas8 or the empty vector. Cell lysates were IPed with anti-FLAG antibody (left two lanes) or run on a gel without immunoprecipitation (right two lanes). Samples were probed with anti-myc and anti-FLAG antibody. Right: IP with anti-myc antibody and IB with anti-FLAG and anti-myc antibody. The results showed that NS1 could be precipitated with Gas8 and Gas8 could also be precipitated with NS1.

Figure 2

Protein-protein interaction domains. (A) Mapping of the NS1 domain interacting with Gas8. 293FT cells were transfected with pCMV-Tag 2B-Gas8 and one of its truncated derivatives: pCMV-myc-NS1_1-80, pCMV-myc-NS1_81-238, or pCMV-myc. After incubation for 36 h, cell lysates were IPed with anti-myc antibody and probed with anti-FLAG and anti-myc antibody. (B) Schematic representation of Gas8 proteins. Polypeptides corresponding to Gas8_1-478, Gas8_1-260, and Gas8_260-478 are shown. The corresponding amino acids are indicated on the right. 293FT cells were transfected with pCMV-myc-NS1 and one of its truncated derivatives: pCMV-Tag 2B-Gas8_1-260, pCMV-Tag 2B-Gas8_260-478, or pCMV-Tag 2B. Cell lysates were IPed with anti-myc antibody and probed with anti-FLAG and anti-myc antibody. +: protein-protein interaction; -: no protein-protein interaction.

Figure 3

Confocal micrographs of NS1 and Gas8 in 293FT cells. Panels (A) and (G): GFP. Panels (D) and (H): DsRed. Panels (B), (E), (I): DAPI. Panels (C), (F), (J): merged images. Top row: cells transfected with pEGFP-NS1 showing nuclear localization. Middle row: cells transfected with pDsRed-GAS8 showing juxtanuclear localization. Bottom row: co-expression of NS1 and Gas8 showing juxtanuclear localization.

Figure 4

Interaction between Gas8 and β-actin. (A)SDS-PAGE analysis of proteins that co-IPed with Gas8. 293FT cells were transfected with pCMV-Tag 2B-GAS8 or pCMV-Tag 2B, and cell extracts were immunoprecipitated with anti-FLAG antibody. The gels were Coomassie stained. 1: pCMV-Tag 2B/293FT, 2: pCMV-Tag 2B-GAS8/293FT. Arrows indicate proteins that co-IPed with Gas8. (B) Gas8 co-localizes with β-actin at the plasma membrane. CV-1 cells were transfected with pDsRed-GAS8. After 24 h, the cells were fixed and probed with an antibody against β-actin. The merged image shows co-localization of Gas8 (red) and β-actin (green) at the plasma membrane. (C) Interaction of Gas8 and β-actin proteins in vitro. 293FT cells were transfected with pCMV-Tag 2B-GAS8 (FLAG-Gas8) or pCMV-Tag 2B (FLAG). Soluble cell lysates were IPed with anti-FLAG or anti-β-actin antibody and probed with anti-FLAG (left) or anti-β-actin antibody (right).