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

293FT, Hela, CV-1, NIH3T3, and BHK21 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated fetal calf serum (HyClone). A549 cells were maintained in McCoy's 5A medium supplemented with 10% heat-inactivated fetal calf serum.

To generate the NS1 expression construct for the CytoTrap two-hybrid system, cDNAs encoding the NS1 proteins of A/Swine/Colorado/1/77 (H3N2) were amplified using the primers listed in Table 1 and cloned into pSos, creating pSos-NS1. To generate an N-terminally myc-tagged NS1 expression construct, the open reading frames encoding NS1 were amplified using the primers listed in Table 1. The PCR products were cloned into pCMV-Myc via the SalI and NotI restriction sites to create pCMV-Myc-NS1. The expression plasmids pGEX-6P-1-NS1 and pEGFP-N3-NS1 were generated by inserting an NS1 cDNA corresponding to amino acids 1 to 237 between the EcoRI/NotI sites of pGEX-6P-1 and the EcoRI/KpnI sites of pEGFP-N3 (NEB), respectively. The GAS8 gene purchased from Proteintech was cloned by PCR using a primer pair specific for the human cDNA (GeneBank accession no. NM_001481). The products were cloned into pDsRed-Express-C1 and pCMV-Tag 2B respectively, creating pDsRed-GAS8 and FLAG-tagged GAS8. Different NS1 and Gas8 deletion mutants were generated by PCR, as above. All primers are listed in Table 1 and all constructs were confirmed by sequence analysis.

The CytoTrap two-hybrid system (Stratagene) was used to identify and isolate CytoTrap XR premade library (Merck) cDNAs encoding NS1 binding factors, according to the manufacturer's instructions. In brief, the CDC25H(α) yeast strain was transformed with the bait plasmid pSos-NS1 and the library DNA purified from the premade libraries, in which human lung cDNAs were conditionally expressed as fusions with a myristoylation membrane localization signal from a GAL1 promoter. A total of 1.09×10⁷ primary transformants were screened for an interaction, as determined by their ability to grow on minimal synthetic medium in the absence of leucine and uracil in plates containing glucose but not galactose at 23°C. Putative clones were isolated by their ability to grow on minimal synthetic medium in the absence of leucine and uracil in plates containing galactose but not glucose at 37°C. The interaction was verified by retransformation of pSos-NS1 with pMyr cDNA plasmid DNA isolated from the putative clones previously identified.

The NS1 gene from avian influenza virus A/Swine/Colorado/1/77 (H3N2) was kindly provided by ShuZhang Feng. 293FT cells were transfected with pDsRed-GAS8 and pDsRed-GAS8/pEGFP-NS1 using Lipofectamine 2000 (Invitrogen). After twenty-four hours, cells were fixed with 4% paraformaldehyde and washed with PBS. Nuclei were stained with DAPI (Sigma). The subcellular localization of the pDsRed-GAS8 and pEGFP-NS1 fusion proteins was detected using a Zeiss LSM 510 META microscope equipped with a 100× objective lens (Zeiss).

293FT Cells were transfected with pCMV-Tag 2B-Gas8 and pCMV-Myc-NS1 expression plasmids. Following transfection for 48 h, the cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris-HCl, pH 7.5) containing protease inhibitors. Lysates were immunoprecipitated (IP) using anti-FLAG M2 (or anti-Myc) mouse monoclonal antibody (Sigma). Immunoprecipitated proteins were immunoblotted (IB) with anti-myc (or anti-FLAG) antibody. Positive bands were detected with the ECL western blotting detection reagent (Amersham), and the image was visualized with CL-X posure™ film (Pierce). To identify the immunoprecipitated Gas8-interacting proteins, 293FT cells were transfected with pCMV-Tag 2B-Gas8 or pCMV-Tag 2B. Following transfection for 48 h, the cells were lysed in RIPA buffer. Lysates were IPed using anti-FLAG M2 mouse monoclonal antibody, and the immunoprecipitated proteins were resolved by SDS-PAGE and Coomassie stained. The peptide mass fingerprint analysis was performed by the National Center for Biomedical Analysis, Beijing.

CV-1 cells were transfected with pDsRed-GAS8. After 24 hours, the cells were washed with 1 ml of PBS and fixed in methanol:acetone (v/v = 1:1). After PBS washes, rat anti-β-actin serum (Sigma) was applied at a dilution of 1:200, followed by a one-hour incubation at room temperature. The cells were washed with PBS and incubated with fluorescein-conjugated affinipure goat anti-mouse IgG (Zhongshan Goldenbridge Biotechnology Co., China) at a dilution of 1:50. Cells were washed with PBS and examined with a confocal microscope.

The CytoTrap system makes use of the yeast S. cerevisiae temperature-sensitive mutant strain cdc25H. The strain grows normally at 23°C, but does not grow at 37°C. The CytoTrap system is based on the ability of the human Sos protein, which is the human homologue of the yeast CDC25 gene, to complement the cdc25 defect and to activate the yeast Ras signaling pathway. Expression of hSos and its subsequent localization to the plasma membrane allows the cdc25H yeast strain to grow at 37°C. The localization of Sos to the plasma membrane occurs through the interaction of two-hybrid proteins. We used a pSos-NS1 fusion protein to screen a human lung cDNA plasmid library, in which cDNA-encoded proteins were conditionally expressed as translational fusions with a pMyr. Expression of the pMyr fusion proteins is induced in the presence of galactose and repressed by glucose. Twenty-four library plasmids were isolated from 1.09 × 10⁷ transformants based on their ability to grow on minimal synthetic medium in the absence of leucine and uracil in plates containing galactose but not glucose at 37°C. Further identification of putative positive plasmids was performed by co-transformation with pSos-NS1, and 15 out of 24 were confirmed as positive plasmids. After DNA sequencing, 8 clones of different gene were obtained. Here, we report the analysis of one of the human cDNAs isolated from these library plasmids that encodes growth arrest-specific gene 8.

To confirm the interaction between NS1 and Gas8 suggested by the two-hybrid results, two experiments were performed. First, GST pull-down assays we performed to find proteins that bind to a GST-NS1 protein in 293FT cell lysates expressing myc-tagged Gas8. Gas8 was efficiently precipitated by a GST-NS1 fusion protein but not by GST (Fig. 1A), indicating that NS1 interacts with Gas8 in vitro. Next, the interaction of NS1 and Gas8 in vivo was investigated by co-immunoprecipitation (co-IP). When lysates of 293FT cells expressing FLAG-tagged Gas8 were immunoprecipitated with anti-FLAG antibodies, myc-tagged NS1 protein was detected in the precipitate. Similarly, Gas8 protein could also be precipitated by NS1 (Fig. 1B).

To identify the region of NS1 protein involved in the interaction with Gas8 protein, in vivo binding experiments were carried out. 293FT cells were transfected with the plasmids pCMV-Tag 2B-Gas8 and pCMV-myc-NS1_1-238 or one of its truncated derivatives: pCMV-myc-NS1_1-80, pCMV-myc-NS1_81-238, or pCMV-myc. When lysates of 293FT cells transfected with the different plasmids mentioned above were immunoprecipitated with anti-myc antibodies, no FLAG-tagged Gas8 protein was detected in the cells expressing truncated NS1 (Fig. 2A). The protein-protein interaction domain of Gas8 was identified by IP assays, as described above, this time using Gas8 deletion mutants. Gas8_1-260 interacted with NS1. In contrast, no interaction with Gas8_260-478 was observed (Fig. 2B).

Based on the two-hybrid and in vitro binding results, we hypothesized that Gas8 and NS1 interact in vivo. If this interaction is physiologically relevant, we would expect the two proteins to occupy the same intracellular compartment. To test this prediction, a Gas8-DsRed and an NS1-GFP fusion were co-expressed in 293FT cells. The Gas8 protein has been reported to localize to the Golgi apparatus [21]. The NS1 protein has been found in large foci in the nucleus [23, 24], a localization that was confirmed in 293FT cells (Fig. 3). When both proteins were co-expressed, fluorescently tagged Gas8 and NS1 exhibited juxtanuclear co-localization. These results suggest that Gas8 can modify the location of NS1.

To find cellular proteins that interact with Gas8, co-immunoprecipitated proteins were identified by peptide mass spectrometry (Fig. 4A). Peptide fingerprinting identified the proteins myosin-9, drebrin E2, and actin. The actin-binding protein drebin has been localized to the apical plasma membrane together with a pool of submembranous F-actin, which is hypothesized to modulate actin-myosin interactions in dendritic spines [25, 26]. Myosin 9 may function in intracellular vesicle transport [27]. CV-1 cells transfected with pDsRed-GAS8 were analyzed by confocal microscopy and immunofluorescence using anti-β-actin antibody. The results showed that Gas8 and β-actin co-localize at the plasma membrane, but not in the cytoplasm (Fig. 4B). Their association was confirmed by co-IP (Fig. 4C).