Cells and viruses.
Parent MDCK cells and plasmid-transfected MDCK cells were maintained in Eagle's minimum essential medium supplemented with 5% fetal bovine serum (FBS). COS-7 cells and plasmid-transfected COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS. IAVs [A/WSN/33 (H1N1), A/Memphis/1/71 (H3N2), and A/duck/313/4/78 (H5N3)] were propagated in 10-day-old embryonated hen's eggs for 2 days at 34°C and were purified by sucrose density gradient centrifugation as described previously (36
Mouse antisulfatide MAb (GS-5; immunoglobulin M [IgM]) (5
) and mouse antiglycosphingolipid, Gb3
Cer MAb (TU-1; IgM) were prepared as described previously (14
). Mouse anti-NP (4E6), anti-H3 HA (2E10 and 1F8), and anti-N2 NA (SI-4) MAbs (IgG) were established by a procedure described previously (20
) using influenza virus A/Memphis/1/71 (H3N2) and A/Japan/305/57 (H2N2) strains. In experiments on virus infection and replication, each MAb was used in the supernatant of each mouse hybridoma cultured with a serum-free medium, Hybridoma-SFM (Invitrogen Corp., Carlsbad, CA).
Cloning and transfection.
Total RNA of cells was extracted with the TRIzol reagent (Invitrogen Corp., Carlsbad, CA) and was converted to cDNA by using a TaKaRa RNA PCR kit (avian myeloblastosis virus), version 3.0 (Takara Bio Inc., Shiga, Japan). The CGT and CST genes from MDCK cells were determined using a 3′-Full RACE Core set (Takara Bio Inc., Shiga, Japan) and a 5′-Full RACE Core set (Takara Bio Inc., Shiga, Japan). We obtained two patterns of open reading frame (ORF) sequence for the CST mRNA from MDCK cells. One had the same number of base pairs as the human CST ORF, but the other had an insertion of 3 bp between positions 131 and 132 relative to the start codon. The CST gene was amplified with PCR primers 5′-CGGAATTCCGATGCCGCTGCCGCAGAAGAAGC-3′ and 5′-CCGGAATTCCGGTCACCACCTCAGAAAGTCCCGGATGA-3′, containing an EcoRI site using pfuUltra high-fidelity DNA polymerase (Stratagene, CA). Two pGEM-CST vectors containing no insertion or a three-base insertion in the CST gene were generated by insertion of the PCR fragment of the CST gene treated with EcoRI into the EcoRI site of the pGEM-T Easy vector (Promega, Madison, WI). The fragment of the CST ORF from pGEM-CST treated with EcoRI was inserted into the EcoRI site of the pIRES-neo vector (BD Biosciences, CA). The CGT gene was amplified with PCR primers 5′-CGGCGGCGGCGTCTCGCATGAAGTCTTACACTCCGTATTCATGC-3′ and 5′-CGGCGGCGTCTCGAATTTTTCACCTTCTTTTCATGTTTAATATGGC-3′ using Tbr EXT DNA polymerase (Finnzymes Oy, Finland). A pTARGET-CGT vector was generated by insertion of the PCR fragment of the CGT gene into the pTARGET vector (Promega, Madison, WI) by TA cloning. A PCR fragment containing two NotI sites was obtained by PCR using the pTARGET-CGT vector as a template with primers 5′-ATAAGAATGCGGCCGCTAAACTATATGAAGTCTTACACTCCGTATTTCATGCTCCTG-3′ and 5′-ATAAGAATGCGGCCGCTAAACTATTCATTTCACCTTCTTTTCATGTTTAA-3′. The PCR fragment of the CGT ORF treated with NotI was inserted into the NotI site of the pIRES-neo vector containing the CST ORF. Then, two bicistronic expression pIRES-CST-CGT vectors (no insertion or a three-base insertion in the CST gene), simultaneously expressing both the CST and CGT proteins from the same mRNA, were generated.
The ASA gene from HeLa cells was amplified with PCR primers 5′-GGAATTCCATGGGGGCACCGCGGT-3′ and 5′-CGGAATTCCGTCAGGCATGGGGATCTGGGC-3′, containing an EcoRI restriction site, using pfuUltra high-fidelity DNA polymerase. A pGEM-ASA vector was generated by insertion of the PCR fragment into the EcoRI site of the pGEM-T Easy vector. The PCR fragment of the ASA ORF amplified with PCR primers 5′-CATGCCATGGGGGCACCGCGGTC-3′ (containing an NcoI restriction site) and 5′-CGGAATTCCGGGCATGGGGATCTGGGCA-3′ (containing an EcoRI restriction site and stop codon deletion due to fusion with a histidine tag) from pGEM-ASA was inserted into the region between the NcoI site and the EcoRI site of the expression pTriEx3-neo vector (Novagen, Darmstadt, Germany). A bicistronic-expression pTriEx3-ASA vector simultaneously expressing both the ASA and neomycin phosphotransferase proteins from the same mRNA was then generated. Sequences of the inserted genes in all generated plasmids were confirmed using an ABI Prism 310NT genetic analyzer (Applied Biosystems, CA).
COS-7 cells were transfected with the pIRES-CST-CGT vector (containing no insertion or a three-base insertion in the CST gene) using the transfection reagent TransIT-293 (Panvera, Madison, WI). The transfected cells were selected in the presence of 1 mg/ml G418 (Promega, Madison, WI) for more than 3 weeks, followed by cloning. Screening of the sulfatide-enriched cell clone was performed by observation of expression of sulfatide stained with GS-5 and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM secondary antibody (Sigma-Aldrich Corp., MO) under a fluorescence microscope (Olympus IX70; Olympus, Tokyo, Japan). Two sulfatide-enriched cell clones, one of which had a three-base insertion in the CST gene, were generated, and stable mRNA expression of the CST gene derived from pIRES-CST-CGT within each cell clone was confirmed by PCR with primers 5′-AGTACTTAATACGACTCACTATAGG-3′ (T7 promoter primer) and 5′-ACGGTGATGAAGGTGGC-3′ (CST antisense primer) using mRNA expression of the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) as a control housekeeping gene (Fig. ).
FIG. 2. Multiple replications of IAV in sulfatide-enriched COS-7 cells. Two sulfatide-enriched cell clones were generated by transfection of COS-7 cells with both dog CST (dCST) and CGT genes and cloning. (A) Detection of dCST mRNA expression in sulfatide-enriched (more ...)
MDCK cells were transfected with the pTriEx3-ASA vector using the transfection reagent TransIT-293. The transfected cells were selected in the presence of 1 mg/ml G418 for more than 3 weeks. Screening of ASA activity and sulfatide depletion in the transfected cells was performed. Sulfatase activities in 120 to 150 μg of the transfected cells were measured as described previously (43
) using 4-methylumbelliferyl sulfate (Research Organics, OH). Sulfatide depletion in the transfected cells was observed by staining of sulfatide with GS-5 and FITC-conjugated goat anti-mouse IgM secondary antibody under a fluorescence microscope. Stable mRNA expression of the ASA
gene derived from pTriEx3-ASA was confirmed by PCR with primers 5′-AGTACTTAATACGACTCACTATAGG-3′ (T7 promoter primer) and 5′-AGCCAGGACTTCGGC-3′ (ASA
antisense primer) using mRNA expression of GAPDH
as a control housekeeping gene (Fig. ).
FIG. 3. Decreased multiple replications of IAV in sulfatide knockdown MDCK cells. Sulfatide knockdown cells (ASA) were generated by transfection of MDCK cells (Parent) with the human ASA gene (hASA) and cultured in the presence of G418 for more than 3 weeks. (more ...) RNAi against CST mRNA.
cDNA sequences 5′-GCTTCAACATCATCTGCA-3′ (CST401-419, numbering from the ATG codon) or 5′-CGACTTCGACTACCCGGCC-3′ (CST337-357) of the CST coding region and 5′-CTGGAGTTGTCCCAATTCT-3′ (GFP29-47) of the green fluorescent protein gene (GFP) coding region were inserted into the cloning site between BamHI and HindIII of the small interfering RNA (siRNA) expression vector pSilencer3.1-H1 neo (Ambion Inc., Austin, TX). MDCK cells were transfected with these vectors using TransIT-293 (Panvera, Madison, WI) and maintained in the presence of G418 (1 mg/ml) for 3 weeks. Then, G418-resistant MDCK cells were cloned in the presence of G418 (500 μg/ml). Endogenous mRNA of the CST gene in each MDCK clone was quantitated by quantitative real-time reverse transcription-PCR (RT-PCR) (LightCycler 2.0; Roche Applied Science, Tokyo, Japan) using the primer pairs 5′-AGCACGGGCTCAAGTTC-3′ (CST302-318) and 5′-ACGGTGATGAAGGTGGC-3′ (CST469-485), which amplify the cDNA region of CST containing CST401-419 and CST337-357. Endogenous mRNA of the GAPDH gene, as a housekeeper gene, was quantitated using the primer pairs 5′-TCAACGGATTTGGCCGTATTGG-3′ (GAPDH17-38) and 5′-TGAAGGGGTCATTGATGGCG-3′ (GAPDH97-106). The relative amount of mRNA of the CST gene is shown as a percentage of that in parent cells. Standard deviations were calculated from the three independent experimental data. To investigate whether RNA interference (RNAi) against the CST mRNA inhibits nuclear export of viral NP, SulCOS1 cells and MDCK cells were transfected with an siRNA vector, piGENE-tRNA Pur (iGENE Therapeutics, Inc., Ibaraki, Japan), in which 5′-GCTTCAACATCATCTGCAACCACATGCGCT-3′ (CST401-430) or 5′-CAACCCGCAGGTGCAGGAACACATCCTGGA-3′ (CST693-722) was inserted between SacI and KpnI sites against CST mRNA of MDCK cells. An siRNA vector in which the GFP sequence 5′-ACTGGAGTTGTCCCAATTCTTGTTGAATTA-3′ was inserted was used as a negative control. The transfected cells were maintained in a serum-free medium for 3 days at 37°C and infected with IAV at a high multiplicity of infection (MOI). After 7 h at 34°C, the infected cells were fixed with cold methanol and stained by 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI), GS-5, and anti-NP (4E6). The stained cells were observed under an LSM 510 confocal microscope (Carl Zeiss Inc., Thornwood, NY).
Virus infection and multiple replications.
Cells were seeded in a 24-well plate (0.5 × 105
cells/well). Cells were infected with IAV strain A/WSN/33 (H1N1) sustained in a serum-free medium at 34°C for 1 h. To examine initiation of infection of IAV, infected cells were maintained in a serum-free medium containing zanamivir (1 μM), a specific IAV sialidase inhibitor, to prevent multiple viral replications. To evaluate multiple viral replications, infected cells were maintained in 500 μl of a serum-free medium without any antibodies or with GS-5 (300 μg/ml) or TU-1 (500 μg/ml) at 37°C in the presence of acetylated trypsin (2.5 μg/ml), which cleaves viral HA to the HA1 and HA2 subunits, in order to activate viral fusion activity, which is required for multiple virus replications. The infected cells were fixed with cold methanol for 30 s, and they were incubated with anti-NP MAb (4E6) for 30 min and then with horseradish peroxidase-conjugated goat anti-mouse IgG plus IgM (Jackson Immuno Research, West Grove, PA) for 30 min at room temperature. Viral NPs within the infected cells were developed by addition of H2
-phenylenediamine dihydrochloride, and 4-chloro-1-naphthol as described previously (36
). Titers of progeny virus in the supernatant from infected cells were measured by a plaque assay. All pictures were taken by using an Olympus DP70 or C5050 camera.
Plaque assay and virus titration.
For quantitation of virus titers, confluent monolayer MDCK cells were incubated with log dilutions of the virus in a serum-free medium for 1 h at 34°C. The infected monolayers were then overlaid with a solution of a serum-free medium containing acetylated trypsin (2.5 μg/ml) and 0.8% agarose. The monolayers were incubated for 2 to 3 days at 34°C until plaques could be visualized. Standard deviations were calculated from the three independent experimental data.
Fluorescence-activated cell sorter analysis of sulfatide.
MDCK cells, sulfatide knockdown MDCK cells, COS-7 cells, and sulfatide-enriched COS-7 cell clones (0.5 × 105 cells/well in a 24-well plate) were maintained in a serum-free medium, Hybridoma-SFM, for 2 days at 37°C and were harvested with treatment of 0.125% trypsin. The cells were fixed with 3% paraformaldehyde for 30 s, and they were incubated in the medium containing GS-5 for 1 h on ice and then with FITC-conjugated goat anti-mouse IgM antibody (Sigma-Aldrich Corp., MO) for 1 h on ice. Fluorescence for cells was excited with the 488-nm line of an argon laser on a fluorescence-activated cell sorter Canto II flow cytometer (BD, Franklin Lakes, NJ). At least 1 × 104 cells were analyzed for each sample.
To visualize sulfatide expression in cells, cells grown on glass coverslips (Teflon printed glass slides; Erie Scientific Company, Portsmouth, NH) were fixed and permeabilized with cold methanol for 30 s, incubated with GS-5, and then incubated with FITC-conjugated goat anti-mouse IgM secondary antibody. To visualize distribution of viral NP, HA, or NA within infected cells, cells were infected with IAV strain A/Memphis/1/71 (H3N2) at an MOI of 5 PFU per cell for 1 h at 34°C and maintained in a medium supplemented with 1% FBS at 37°C. At 7 h postinfection, the infected cells were fixed and permeabilized with cold methanol for 30 s. The infected cells were incubated with mouse anti-NP (4E6), anti-H3 HA (2E10), or anti-N2 NA (SI-4) MAb and then with tetramethyl rhodamine-conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich Corp., MO). Nuclei were visualized with DAPI (Dojindo Laboratories, Kumamoto, Japan). The cells were observed under a confocal microscope. To evaluate nuclear localization of NP in sulfatide-enriched or knockdown cells, pictures (magnification, ×400) with staining of NP and nuclei were taken at 5.5 and 6.5 h postinfection at 34°C. Cells in which most of the NP was localized in the nucleus were counted in least six fields per cell line. Nuclear localization of NP in each cell line was expressed as the ratio of cells with nuclear localization of NP to total infected cells.
Inhibition of IAV binding to sulfatide by MAbs.
Five hundred picomoles of sulfatide was immobilized in wells of microtiter plates (F96 Polysorp; Nunc, Roskilde, Denmark) as described previously (37
). Each well was blocked by 0.2% bovine serum albumin containing 0.1% (wt/vol) MEGA-10 (Dojindo Laboratories, Mashiki, Japan) at 4°C overnight. For the binding inhibition test of anti-IAV MAbs (2E10, 1F8, and SI-4), 50 μl of influenza virus A/Memphis/1/71 (H3N2) suspension (24 hemagglutinating units) was treated with 50 μl of each anti-IAV MAb at 4°C for 2 h. The reaction mixtures were added to the well and incubated at 4°C for 2 h. For the binding inhibition test of anti-glycolipid MAbs (GS-5 and TU-1), 50 μl of each MAb was added to the wells. After a 2-h incubation at 4°C, 50 μl of IAV suspension (24 hemagglutinating units) was added to the wells and incubated at 4°C for 2 h. Binding of virus to sulfatide was detected by using rabbit anti-IAV polyclonal antibody and horseradish peroxidase-conjugated protein A as described previously (37
in vivo antiviral assay.
Female C57BL/6 mice (8 weeks) were infected intranasally with 20 μl of a 4 × 103 50% tissue culture infective dose of mouse-adapted IAV A/WSN/33 (H1N1). Twenty-five microliters of phosphate-buffered saline containing GS-5 (300 μg/ml) was administered intranasally once daily for 5 days beginning 1 day preinfection. Four mice per group were monitored for body weight and mortality daily for 7 days after infection until all mice had succumbed to infection or were recovering as assessed by body weight. Lung histopathology was examined on day 6 after IAV infection.
Nucleotide sequence accession numbers.
Nucleotide sequence data for the dog CST genes are available in GenBank under accession numbers 240641 and 240642.