2.1. Cells and viruses
Human embryonic kidney 293 (HEK293) and HeLa cells were used in transient transfection experiments and/or viral infections. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (Gibco, USA), 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 1.5 mg/ml sodium bicarbonate at 37°C in a humidified incubator with 5% CO2. Avian influenza virus A/duck/Pennsylvania/10,218/84/H5N2 (DkPen) were propagated in specific pathogen-free chicken embryos. The viral titer was measured using a standard plaque-forming assay [30].
2.2. RNA extraction and first-strand cDNA preparation
For PCR amplification of SNX2 cDNA and/or quantitation of transcripts with a real-time polymerase chain reaction (qPCR), total RNA was extracted from the cells with the RNeasy mini kit (Qiagen, Germany). cDNAs were prepared from the total RNA by Moloney murine leukemia virus reverse transcriptase (ReverTra Ace, Toyobo Co. Ltd., Japan) and oligo dT as a primer for 60 min at 45 ºC.
2.3. Construction of plasmid vectors
For the construction of Sorting Nexin 2 (SNX2) expression plasmids, the full length of the SNX2 cDNA consisting of 1560 bp was amplified by PCR with HEK293 cDNA and the phosphorylated specific primers, 5'-ATCATGGCGGCCGAGAGGGAA-3' and 5'-ATCTAGGCAATGGCTTTGGCTTC-3'. PCR amplification was carried out with a thermostable DNA polymerase (KOD plus, Toyobo Co., Ltd., Japan). The PCR product was purified with an agarose gel extraction kit (QiaexII, Qiagen, Germany). In order to construct the pCHA-SNX2 plasmid encoding the HA-tagged SNX2 protein (H.SNX2), the SNX2 cDNA was cloned into a pCHA [31] plasmid digested with EcoRV (New England Biolabs, UK) and dephosphorylated with Shrimp Alkaline Phosphatase (Thermo Fisher Scientific, USA). The plasmids encoding deleted SNX2 proteins were constructed with the inverse PCR method by using the pCHA-SNX2 plasmid as a template. For SNX2ΔN (exons 1–3 deleted), 5'-GATATCACGCGTGGTGACC-3' and 5'-ATGATTGAAGAAGAAGCAAATGG-3' were used; for SNX2ΔM (exons 4–8 deleted), 5'-CTCTTCCCTGGATCTATCAAAG-3' and 5'-CTGCCTAGAGCAGTTAATACAC-3' were used; for SNX2ΔC (exons 9–15 deleted), 5'-TAGATATCTTAAGTGACTGAATTC-3' and 5'-CTCTGAACTTTCCAAGAACTG-3' phosphorylated primer pairs were used. The PCR products were purified with an agarose gel extraction kit and self-ligated.
In order to construct the bait plasmid coding influenza A/DkPen PA protein (pGBD-PA), the DNA fragment was amplified with PCR by using virus-infected cell cDNA as a template and the phosphorylated specific primers, 5'-CGGAGGATCTGGAATGGAAGACTTTGTGCGACAATG-3' and 5'-CTAGTTCTTTGTCTTTGGGATCTTC-3'. The PCR amplified gene was ligated with the pGBD-C1 [32] plasmid DNA digested with EcoRI and blunted with Klenow enzyme. For the construction of the pACT2-SNX2 yeast two-hybrid plasmid, the PCR amplified full-size SNX2 cDNA was ligated with pACT2 (Clontech, # 638822) plasmid digested with BglII and blunted with Klenow enzyme. The nucleotide sequences of all plasmids were confirmed by DNA sequencing. The plasmids for expression of viral PA proteins in mammalian cells have been described previously [33, 34].
2.4. Yeast two-hybrid screening
Saccharomyces cerevisiae strain PJ69-4A was grown in 5 ml YPAD media at 30 °C in a shaking incubator at a speed of 200 rpm overnight. The fresh culture was transformed with pGBD-PA bait plasmid coding GAL4.BD-PA fusion by using the lithium acetate/polyethylene glycol (LiAc/PEG) protocol [35]. The transformants were selected on a synthetic defined (SD) agar medium (without Trp). One of the bait colonies was grown in YPAD media and re-transformed with a cDNA library derived from HEK293 cells (Clontech # 638826) and screened following the matchmaker two-hybrid system protocol. The positive transformants were selected on the SD agar medium (without adenine, histidine, leucine, and tryptophan) and then tested by the β-galactosidase assay for the second screening. The plasmids carrying the cDNAs were isolated from the transformants with a yeast plasmid DNA miniprep kit (Bio Basic, Canada) according to the manufacturer’s instructions, and transformed E. coli DH5α and amplified. The cDNA inserts in the plasmids were sequenced and identified with BLAST (Basic Local Alignment Search Tool) analysis.
2.5. Transforming pACT2-SNX2 into the yeast cells and checking SNX2 and PA interaction
pACT2-SNX2 or pACT2 plasmid (control) were transformed into the S. cerevisiae strain PJ69-4A harboring the bait plasmid (pGBD-PA) with LiAc/PEG protocol. Double transformants were selected on SD agar medium (without Trp and Leu). A few colonies were cultured on a SD agar medium (without His, Leu, Ade, and Trp); growth profiles, were defined and tested for β-galactosidase activities.
2.6. β-galactosidase assay
Transformed yeast cells were grown in 5 ml SD medium (without Trp/Leu or Leu) at 30 ºC. The cells in 500 µl of saturated culture were recovered with centrifugation and re-suspended in 300 µl Z-buffer (0.1 M sodium phosphate, pH 7.0, 10 mM KCl, 1 mM MgSO4 and 0.27% β mercaptoethanol). The cells in suspensions were disintegrated with the freeze-thaw procedure in liquid nitrogen. Then, the samples were mixed with 60 µl o-nitrophenyl-β-d-galactopyranoside (ONPG) (4 mg/ml) and incubated for 60 minutes at 37 ºC. Three hundred µl Na2CO3 (0.5 M) was added to stop the reactions. The supernatants were recovered by centrifugation at 15000 rpm for 5 min and the absorbance of the samples at 420 nm (OD420) was defined.
2.7. Plasmid DNA transfection
Polyethylenimine (PEI) was used for plasmid DNA transfection into the HEK293 or HeLa cells (PA Longo et al. 2013). Plasmid DNA and PEI were diluted in OPTI-MEM (Gibco, USA) to a concentration of 20 ng/µl and 40 ng/µl, respectively, and mixed in equal volumes to form complexes. After incubation at room temperature for 5–10 minutes, the complexes were added to the culture media. The cultures were incubated under the standard culture conditions for 20–48 hours and used in the experiments.
2.8. Immunoblotting
The expression of the SNX2 proteins and viral proteins in transfected and/or virus-infected cells were analyzed with western blotting. The cells grown in 12-well plates were lysed in lysis buffer A (50 mM Tris- HCl pH 8.0, 150 mM NaCl, 1 mM DTT, and 0.1% NP-40) or SDS-PAGE sample buffer. The proteins were separated using SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, the membrane was exposed to the specific primary antibodies (monoclonal mouse anti-HA [Santa Cruz # sc-7392]; monoclonal mouse anti-actin [Mybiosource # MBS9400413] or anti-PA polyclonal rabbit antisera overnight at 4°C and then to the horseradish peroxidase-conjugated second antibody (anti-mouse IgG-HRP [Invitrogen # 31420] and/or anti-rabbit IgG-HRP [Invitrogen # 31423]) against species-specific immunoglobulins for 45 minutes at room temperature. The proteins were visualized with an ECL detection kit (GE Healthcare, Italy).
2.9. Co-immunoprecipitation assay
Co-immunoprecipitation assays were carried out to confirm the interaction between PA and SNX2 proteins in mammalian cells. The HEK293 cells were seeded in a 6-well plate (5x105 cells/well) and incubated at 37°C in a humidified incubator with 5% CO2 for 20–24 h. The cells were co-transfected with the plasmid DNAs expressing SNX2 and viral PA proteins and incubated for 48 h. After the incubation periods, the cells were washed with PBS and treated with 1 mM DSP (dithiobis [succinimidyl propionate]) for 1 h. The cross-linking reaction was stopped by the addition of glycine at 100 mM final concentration, and then the cells were lysed in lysis buffer A. The cell lysates were clarified by centrifugation at 10000 rpm for 5 min. The lysates (250 µL) were incubated with a 5 µL monoclonal mouse anti-HA antibody (sc-7392) at 4°C for 2 h. Protein A-Sepharose (GE Healthcare, Sweden) was added to the lysates and moderately stirred overnight at 4°C. The beads were washed three times with buffer A. The proteins bound Sepharose beads were recovered by heating the samples at 95°C for 5 minutes in a SDS-PAGE sample buffer and then analyzed by western blotting.
2.10. Immunofluorescence assay
The localization of H-SNX2 and influenza PA proteins in transiently transfected HeLa cells was examined with immunofluorescence staining. The monolayers of HeLa cell on coverslips were transfected with pCAGGS-PA, pCHA-SNX2, pCHA-SNX2/pCAGGS-PA, pCHA-SNX2/pCAGGS-nPA, or pCHA-SNX2/pCAGGS-cPA plasmids. After 36–40 hours transfection, the cells were washed with PBS; fixed in 3% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% NP-40, washed twice with PBS, and then treated with 1% skim milk for 30 min. The cells were incubated with primary antibodies (mouse anti-HA and rabbit anti-PA) diluted in 1% skim milk for 60 min and washed three times with PBS. The cells were treated with 1% skim milk for 20 min once again and then stained with Alexa-488-conjugated goat anti-mouse IgG and/or Alexa-568-conjugated goat anti-rabbit IgG (at 1:300 dilutions in 1% skim milk) for 60 min. The nuclei of the cells were stained with DAPI. The coverslips were washed with PBS and mounted in a media (0.1% p- phenylenediamine and 80% glycerol) and the cells were analyzed with a laser confocal microscope (Zeiss LSM 700).
2.11. In situ proximity ligation assay (PLA)
The 50–60% confluent HeLa cells grown on coverslips were co-transfected with SNX2 and viral PA coding plasmids. After 36–40 hours of transfection, the cells were treated with primary antibodies (monoclonal mouse anti-HA and polyclonal rabbit anti-PA) as mentioned above. The assay was carried out with a Duolink PLA kit (Sigma-Aldrich # DUO92104) by following the manufacturer’s instructions. Briefly, the monolayers were washed with wash buffer A (10 mM Tris, pH 7.4, 150 mM NaCl and 0.5% Tween-20) for 10 min and treated with a mixture of plus (mouse) and minus (rabbit) PLA probes for 90 min. Then, the monolayers were washed three times with wash buffer A for 10 min and subjected to ligation. After ligation, the closed circles were amplified using a rolling-circle amplification with the DNA polymerase and complementary fluorescently labeled oligonucleotides for three hours at 37 ºC. The samples were washed three times with wash buffer B (200 mM Tris, pH 7.5, 100 mM NaCl) for 10 min, rinsed once with 0.01x wash buffer B, mounted in the mount media, and visualized with the laser confocal microscope (Zeiss LSM 700).
2.12. siRNA transfection, and quantitation of viral RNAs and viral proteins
The small interfering RNA (siRNA) targeting exon 2 of the SNX2 gene (Cat. / Assay ID: Cat#1299001/HSS110066) was purchased from Life Technologies. The HeLa cells were seeded in six cm petri dishes (5x105 cells) and incubated at standard culture conditions for 20–24 hours. The cells were transfected with 30 pmol SNX2 gene-specific siRNA or negative control siRNA (Invitrogen # 12935–200) with lipofectamine RNAiMAX (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol and incubated for 48 h. Then, the cells were sub-cultured into 12-well plates (2x105 cells/well) for 24 h. After the incubation period, total RNA was extracted from some monolayers for quantitation of the SNX2 transcript. Some of the monolayers were infected with influenza A/DkPen viruses at a one moi. After virus adsorption at 37 ºC for 30 min, the inoculum was removed, and the cells were maintained in the maintenance medium (DMEM containing 0.2% Bovine Serum Albumin and 4 mg/ml trypsin) for six or 12 hours. The cells were lysed in SDS-PAGE sample buffer at the end of the incubation period and used for viral protein analysis with western blotting. At eight h post infection, the total RNA was extracted from some of the infected cultures for quantitation of the viral RNAs. Quantitation of SNX2 transcript and viral mRNAs/cRNA (segment 8) was carried out with qPCR. qPCR was conducted using FastStart Universal SYBR Green Master Mix (Roche, Germany). The cycle conditions included an initial denaturation step at 95°C for 10 min, followed by 45 cycles of amplification for five s at 95°C, 10 s at 55–60°C and 20 s at 72°C. The quantities of SNX2 transcript and viral RNAs were normalized by the amount of actin beta (ACTB). The oligonucleotide primers used in the real-time PCR are given in Table 1.
Table 1
The oligonucleotide primers used in qPCR experiments.
Primer name
|
Sequence
|
Product Size
|
SNX2/846 For
|
5'-GAGGATGGTGAACAAGGCTG-3'
|
146 bp
|
SNX2/992 Rev
|
5'-ACCAAGGCTTCAACACTGAC-3'
|
Seg-8/542 For
|
5'-TCATCGGTGGACTTGAATGG-3'
|
144 bp
|
Seg-8/686 Rev
|
5'-TCTGACTCAACTCTTCTCGC-3'
|
Actin/280 For
|
5'-CCACACCTTCTACAATGAGC-3'
|
272 bp
|
Actin/552 Rev
|
5'-TCATGAGGTAGTCAGTCAGG-3'
|
2.13. Titration of influenza A virus PA inhibitory effects on reporter SEAP expression with SNX2 proteins