Human rhinovirus 14 enters rhabdomyosarcoma cells expressing icam-1 by a clathrin-, caveolin-, and flotillin-independent pathway

Abstract

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na(+)/H(+) ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via alpha(v) integrin/CD46 in HeLa cells.

FIG. 1.

HRV14 does not colocalize with transferrin. RD-ICAM cells were incubated with HRV14 at 300 TCID₅₀/cell in the presence of 10 μg/ml Alexa Fluor 568-transferrin for 60 min at 4°C. Unbound material was washed away, and cells were shifted to 34°C. At the times indicated, cells were fixed and stained for virus with type-specific mouse antiserum followed by Alexa Fluor 488-labeled secondary antibody. Colocalization was assessed by confocal fluorescence microscopy. In the lower panels (15 min) 10 confocal slices were combined. Bar, 10 μm.

FIG. 2.

DN inhibitors of the clathrin-dependent pathway have no effect on either entry or replication of HRV14. RD-ICAM cells were transfected with DN myc-tagged amphi-SH3 and AP180-C, as indicated, and challenged with HRV14 at 300 TCID₅₀/cell for 30 min at 34°C (continuous internalization) (A) and at 5 TCID₅₀/cell for 7 h (B). Virus was detected with type-specific antiserum and Texas Red-labeled secondary antibody, whereas expression of the DN inhibitors was assessed by using mouse anti-myc monoclonal antibody, followed by Alexa Fluor 488-labeled secondary antibody; samples were processed and viewed as described in the legend of Fig. 1. One confocal slice through the cell body is shown. Transferrin was used as a control. Bar, 10 μm.

FIG. 3.

HRV14 does not colocalize with CtxB. RD-ICAM cells were incubated with HRV14 at 300 TCID₅₀/cell and 1 μg/ml Alexa Fluor 488-CtxB for 60 min at 4°C. Unbound material was washed away, and cells were shifted to 34°C. At the times indicated, cells were fixed and stained for virus with type-specific mouse antiserum followed by Texas Red-labeled secondary antibody. Colocalization was assessed by confocal fluorescence microscopy as described in the legend of Fig. 1. Bar, 10 μm.

FIG. 4.

Flotillin-1 does not colocalize with HRV14. RD-ICAM cells were infected with HRV14 at 300 TCID₅₀/cell for 5 and 15 min at 34°C (continuous internalization), unbound virus was removed, and the cells were fixed. Flotillin-1 was stained with rabbit anti-flotillin-1 antibody followed by Texas Red-conjugated secondary antibody. Virus was detected with type-specific antibody, followed by Alexa Fluor 488-labeled secondary antibody. Cells were processed and viewed as described in the legend of Fig. 1. One single confocal slice is shown. Bar, 10 μm.

FIG. 5.

Inhibition profile of pharmacological inhibitors. Cells were grown in 24-well plates until 90% confluent. The drugs were added at the concentrations indicated in Materials and Methods and incubated for 30 min. (A) Then, virus was added at 300 TCID₅₀/cell, and incubation continued for another 3 h. After removal of the supernatant, the cells were harvested in sample buffer, and proteins were separated on an SDS-6% polyacrylamide gel. Cleavage of eIF4GI was monitored via Western blotting with a rabbit antiserum, followed by HRP-conjugated anti-rabbit antibodies, and proteins were detected by chemiluminescence recorded on X-ray film. (B) Cleavage was quantified from scans of X-ray films from three independent experiments, including that shown in panel A, by using ImageJ software. Values are means ± standard deviations (SD). Note that the extent of cleavage under control conditions (i.e., absence of inhibitors) differed for the two virus types. (C) Cells were grown on coverslips until 70% confluent, preincubated with 5 mM amiloride and 10 μg/ml cytochalasin for 30 min as indicated, and challenged with HRV14 for 30 min at 34°C. Virus was stained and viewed as described in the legend of Fig. 1 but before (Pre-perm) and after (Post-perm) permeabilization by using secondary antibodies conjugated with Texas Red and Alexa Fluor 488, respectively. Note the strong intracellular staining of HRV14 in the controls (arrows) that is absent from amiloride-treated cells and substantially reduced in cytochalasin-treated cells. (D) For control purposes, the same experiment was carried out with HRV2. Bar, 10 μm.

FIG. 6.

HRV14 colocalizes with FITC-dextran and induces its uptake. (A) RD-ICAM cells were infected with HRV14 at 300 TCID₅₀/cell (continuous internalization) in the presence of 500 μg/ml lysine-fixable FITC-dextran for the times indicated at 34°C, washed, and prepared for immunofluorescence microscopy as described in the legend of Fig. 1. Despite the strong signal of HRV14 because of the ICAM-1 overexpression, colocalization with FITC-dextran is evident. Bar, 10 μm. (B) Subconfluent RD-ICAM cells serum starved for 4 h were incubated with serum-free medium without EGF (no stimulation) or with 100 ng/ml EGF (control stimulation) and 5 μg/ml purified HRV14, together with 1 mg/ml 70-kDa FITC-dextran for 10 min at 37°C. Cells were washed with cold PBS and detached with trypsin at 4°C, and dextran uptake was determined by flow cytometry in a BD FACSCalibur. Percent increase of mean fluorescence of the cells related to the control is shown. Values are the means of three independent experiments ± SD.

FIG. 7.

Electron microscopy of virus entry. RD-ICAM cells grown on coverslips were infected with HRV14 at 10,000 TCID₅₀/cell for the times indicated at 34°C. Cells were fixed and stained with OsO₄, embedded in resin, and cut into 70-nm slices. They were viewed at a magnification of ×35,000. For a control, cell slices depicting HRV2 entry in coated pits and vesicles are shown. Selected areas from the middle panels are shown at higher magnification in the right panels. Note the absence of HRV14 in clathrin-coated vesicles (upper panels), whereas HRV2 is clearly seen in clathrin-coated pits and vesicles (lower panels). Areas of interest are indicated with arrows. Bar, 350 nm.