Miller, L. together, these results show that HRV2 uses clathrin-mediated endocytosis to infect cells. Human rhinoviruses, members of the family (7) was amplified from an MDCK cDNA library (a kind gift of I. Fialka and L. Huber, Institute of Molecular Pathology, Vienna, Austria) and cloned as a myc-tag fusion protein in the same altered pCI vector. The Ser34 Asn mutation was launched by PCR using primer elongation. Internalization of HRV2 and of fluorescent-transferrin conjugates. Cells were produced on coverslips and transfected with plasmids encoding the dominant-negative mutants 2 days before the experiment. They were then incubated with computer Syringin virus (30 PFU per cell) for 20 min at 34C in MEM contamination medium, washed with PBS, and prepared for immunofluorescence. For cointernalization of HRV2 and fluorescent transferrin, cells were preincubated with MEM without FCS at 34C for 30 min. The medium was replaced by MEM contamination medium Syringin without FCS and made up of 5 g of rhodamine-transferrin/ml together with HRV2. Incubation was at 34C for 20 min, followed Syringin by three washes with ice-cold PBS prior to fixation. Fluorescence microscopy. Cells were fixed in 3% paraformaldehyde in PBS for 15 min at room heat, quenched for 10 min in 50 mM NH4Cl in PBS, washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed three times, and blocked with 5% FCS in PBS for 15 min. Incubation with main antibodies (8F5 or HA-probe) diluted 1:200 in PBS made up of 1% FCS was performed for 1 h at room temperature. Cells were washed with PBS and incubated for 1 h at room temperature with secondary antibodies diluted 1:400 in PBS made up of 1% FCS. The coverslips were washed three times with PBS and rinsed briefly in double-distilled H2O, and the cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, Calif.). Samples were viewed under a Leica TCS NT confocal microscope (Heidelberg, Germany). Images were processed using Adobe Photoshop software. Potassium depletion. HeLa-H1 cells were washed with K+-free buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 1 mg of d-glucose/ml [pH 7.4]), incubated with K+-free buffer diluted 1:1 with water (hypotonic buffer) for 5 min, washed three times with K+-free buffer, and incubated with HRV2 for 20 min at 34C in the same buffer prior to fixation and immunofluorescence. As a control, the same buffers made up of 10 mM KCl were used. Cholesterol depletion and virus-uncoating assay. HeLa-H1 cells produced in 24-well plates were preincubated with 10 mM methyl–cyclodextrin (MCD) in MEM contamination medium at 34C for 30 min. For control purposes, cells were also incubated without any addition of MCD or in the presence of Syringin 400 nM bafilomycin A1. HRV2 (30 PFU per cell) was added, and the incubation was continued for 20 min in the absence or presence of the drugs. After being washed three times with PBS, samples were taken immediately (= 0) or after further incubation for the specified time periods prior to determination of the computer virus titer. Computer virus titer determination. Infected cells were broken by three freeze-thaw cycles, debris was removed by a brief centrifugation, and serial 10-fold dilutions of the supernatants were prepared in MEM contamination medium. Samples were transferred onto subconfluent monolayers of HeLa-H1 cells produced in 96-well culture plates made up of 100 l of MEM contamination medium. After incubation at 34C for 5 days, cells were stained with 0.1% crystal violet (in water) for 20 min. The tissue culture infective dose which infects 50% Rabbit Polyclonal to SERPINB9 of the cells (TCID50) was calculated according to the method of Blake and O’Connell (5). RESULTS Because previous reports have suggested internalization of HRV2 via a clathrin-independent Syringin pathway (3, 23, 34) we in the beginning decided to study the underlying mechanism by asking whether LDLRs lacking the coated-pit localization transmission were able to mediate viral contamination. The full-length human LDLR or a mutant lacking 33 amino acids at the C terminus, including the clathrin-coated-pit-targeting signal NPVY, were stably transfected into a fibroblast cell collection (M4) deficient in LDLR and LRP expression as a result of gene disruption (21, 24, 68). Regrettably, the expression level of the truncated LDLR was much higher than that of wild-type.