Vero C1008, Vero E6 (both kindly provided by G. Darai, Heidelberg) and Madin-Darby canine kidney II (MDCKII) (European Collection of Cell Cultures, ECACC) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics. Human renal proximal epithelial cells, HREpC, were obtained from Promocell and maintained in renal epithelial cell growth medium 2 (Promocell). Only HREpCs from passage two to six were used. For polarized monolayers, cells (1×105) were plated on 0.4-μm-pore-size 12 well cell culture inserts (Greiner Bio-One) and cultivated for 7 days.
Virus and infection
The stocks of hantavirus species Hantaan virus, strain 76–118 (HTNV) (kindly provided by G. Darai, Heidelberg) or Puumala virus, strain Vranica (PUUV) (kindly provided by S. Essbauer, Munich) were propagated on Vero E6 cells. To infect cells, virus inocula, HTNV or PUUV, at an MOI of 0.01 were added to Vero C1008, HREpC, or MDCKII cells; after incubation for 1 h at 37°C, unbound virus was removed by a triple washing. Cells were grown in tissue culture dishes containing coverslips. The infection was monitored by the immunofluorescence or by the Western blot analysis of hantaviral nucleocapsid protein expression. Expression of nucleocapsid protein of HTNV and of PUUV was analyzed in all cell types at day 4 and day 8 post infection, respectively. An equal loading was verified by the detection of tubulin on the same membrane. For re-infection, cells grown on coverslips were inoculated with cell-free supernatants of infected cells and monitored for infection.
Immunofluorescence and Western blot analysis
For immunofluorescence, cells grown on coverslips or on cell culture inserts were actone-fixed and stained with primary and appropriate fluorescently labelled secondary antibodies. The following antibodies were used: mouse monoclonal anti-nucleocapsid protein (Progen), rabbit anti-ZO-1 (Invitrogen), mouse anti-β-catenin (Santa Cruz). Images were taken using a Nikon DXM1200C camera attached to a Nikon Eclipse 80i upright microscope (Nikon). Series of optical sections distanced 0.15 μm on the z axis were taken with a Perkin Elmer spinning disc confocal ERS-FRET on Nikon TE2000 inverted microscope.
For Western blot analysis, cells were lysed and after being boiled in SDS sample buffer and separated by SDS-PAGE, transferred to a nitrocellulose membrane. The Western blot analysis was performed after the incubation with primary antibodies by using near infrared fluorescent dye (IRDye)-conjugated secondary antibody and an Odyssey infrared imaging system (Li-Cor Biosciences). The following primary antibodies were used: rabbit polyclonal anti-PUUV or anti-HTNV nucleocapsid protein antibody and mouse anti-α-tubulin DM 1A (Sigma).
For analysis by scanning electron microscopy (SEM), MDCKII cells were fixed with 2.5% (v/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 100 mM cacodylate buffer (pH 7.4) for 30 min at room temperature. After fixation cells were rinsed three times for 10 min with 100 mM cacodylate buffer, and dehydrated through a graded ethanol series. After washing three times with hexamethyldisilazane (Electron Microscopy Sciences) cells were coated with gold and analyzed on a LEO 1430 scanning electron microscope.
For analysis by transmission electron microscopy (TEM), MDCKII cells were fixed with 2.5% (v/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 100 mM cacodylate buffer (pH 7.4) for 30 min. Cells were rinsed three times for 5 min with 100 mM cacodylate buffer, postfixed for 1 h in 1% (v/v) osmiumtetroxide, rinsed three times with distilled water, en bloc stained with 0.5% (v/v) uranyl acetate, dehydrated through a graded ethanol series and finally embedded using EMBed 812 (Electron Microscopy Sciences). Cells were cut perpendicular to the substrate and 70–90 nm sections were collected. Sections were counterstained with 4% (w/v) uranyl acetate followed by lead citrate. All samples were imaged on a transmission electron microscope equipped with a wide-angle CCD camera (Zeiss EM 900, TRS Systems).
For flow cytometry MDCKII cells were washed, scraped and stained with allophycocyanin (APC)-conjugated rabbit polyclonal anti-CD55 antibody, clone IA10 (BD Pharmingen) and phycoerythrin (PE)-conjugated mouse anti-integrin αVβ3antibody, clone LM609 (Millipore). After 1 hour of incubation the cells were washed and then analyzed by flow cytometry with FACSCalibur (BD Pharmingen). Controls were incubated with APC- and PE-conjugated mouse and rabbit isotype antibodies.