Reovirus uses multiple endocytic pathways for cell entry

Abstract

Entry of reovirus virions has been well studied in several tissue culture systems. After attachment to junctional adhesion molecule A (JAM-A), virions undergo clathrin-mediated endocytosis followed by proteolytic disassembly of the capsid and penetration to the cytoplasm. However, during in vivo infection of the intestinal tract, and likely in the tumor microenvironment, capsid proteolysis (uncoating) is initiated extracellularly. We used multiple approaches to determine if uncoated reovirus particles, called intermediate subviral particles (ISVPs), enter cells by directly penetrating the limiting membrane or if they take advantage of endocytic pathways to establish productive infection. We found that entry and infection by reovirus ISVPs was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis, as well as by genistein and dominant-negative caveolin-1, which block caveolar endocytosis. Inhibition of caveolar endocytosis also reduced infection by reovirus virions. Extraction of membrane cholesterol with methyl-β-cyclodextrin inhibited infection by virions but had no effect when infection was initiated with ISVPs. We found this pathway to be independent of both clathrin and caveolin. Together, these data suggest that reovirus virions can use both dynamin-dependent and dynamin-independent endocytic pathways during cell entry, and they reveal that reovirus ISVPs can take advantage of caveolar endocytosis to establish productive infection.

Fig 1

Reovirus virions and ISVPs undergo dynamin-mediated endocytosis. Reovirus T1L virions or ISVPs were adsorbed at a concentration of 1 × 10⁵ particles/cell to adherent A549 cells that had been pretreated with vehicle (DMSO) or dynasore. The monolayers were fixed at 10 min postinfection and labeled with anti-reovirus T1L antiserum (green), phalloidin (red), and DAPI (blue). Scale bars represent 20 μM.

Fig 2

Dynamin plays a role in productive infection by reovirus virions and ISVPs. (A and B) L929 cells were pretreated with vehicle (DMSO) or dynasore and infected with reovirus T1L virions (A) or ISVPs (B) at an MOI of 3. After adsorption, fresh medium with vehicle or inhibitor was added to the cultures, and samples were collected at the indicated times postinfection. Viral yields were quantified by plaque assay on L929 cells. A549 (C) and 293 cells (D) were pretreated with DMSO or dynasore and infected at an MOI of 3. Samples were harvested at 24 hpi, and viral yields were quantified by plaque assay on L929 cells. (E) Dynasore was added to L929 cells at the indicated times prior to or after adsorption with reovirus virions. Infected cell lysates were analyzed at 24 hpi for μNS expression by immunoblotting. Band intensities of μNS and β-actin were quantified to determine the relative μNS expression level of each sample. The values in the graph represent the relative μNS expression level compared to vehicle-treated cells.

Fig 3

Dynasore inhibits pathway-specific ligand uptake but not reovirus binding or cell viability. (A) L929 cells in suspension culture were pretreated with vehicle (DMSO) or dynasore and adsorbed with reovirus virions at a concentration of 1 × 10⁵ particles/cell (white peaks) or an equivalent volume of gel saline (gray peaks). After adsorption, cells were fixed and stained with an anti-reovirus T1L antibody. Reovirus binding was quantified by flow cytometry. (B) A549 cells were pretreated as described above and then incubated with Alexa 488-transferrin or FITC-CTB. After 1 h at 4°C, warm medium was added to the cultures and the cells were fixed after 30 min. The percentage of transferrin- or CTB-positive cells was determined by counting three similarly confluent fields in three independent experiments. Between 450 and 750 cells were counted in each experiment. Error bars represent standard errors of the means for the three independent experiments. (C) L929 cells were treated with dynasore at the indicated concentrations and incubated for 24 h at 37°C. Cells were stained with annexin V/PI, and viability was assessed by flow cytometry. A parallel set of samples was pretreated with dynasore, infected with T1L virions or ISVPs at an MOI of 3, and assayed for viral yield at 24 hpi.

Fig 4

Caveolar endocytosis is important for infection by reovirus virions and ISVPs. (A and B) L929 cells were pretreated with vehicle (DMSO) or 200 μM genistein and infected with reovirus T1L ISVPs (A) or virions (B) at an MOI of 3. Viral yields were determined at the indicated times postinfection by plaque assay on L929 cells. (C) A549 and 293 cells were pretreated and infected as described above, and viral yields were determined at 24 hpi. (D) HeLa cells seeded in 8-well CultureSlides were pretreated as described above and infected at an MOI of 15. At 24 hpi, cultures were fixed and stained with antiserum specific for reovirus T1L. Infection was analyzed by indirect immunofluorescence, and the percentage of infected cells was determined from three equally confluent fields of view. (E) L929 cells were pretreated with the indicated concentrations of genistein, and viability was assessed by annexin V/PI staining at 24 hpi. A parallel set of samples was infected with T1L virions or ISVPs and assayed for viral yield at 24 hpi. (F) A549 cells were pretreated as described above and incubated with Alexa 488-transferrin or FITC-CTB. After 1 h at 4°C, warm medium was added to the cultures and the cells were fixed after 30 min. The percentage of transferrin- or CTB-positive cells was determined by counting three similarly confluent fields. Error bars represent the standard deviations for each sample. (G) Genistein was added to L929 cells at the indicated times prior to or after adsorption with reovirus virions. Infected cell lysates were analyzed at 24 hpi for μNS expression by immunoblotting. The immunoblot (left) is from a representative experiment. Band intensities of μNS and β-actin were quantified to determine the relative μNS expression level of each sample. The values in the graph are derived from three independent experiments and represent the relative μNS expression level compared to that in vehicle-treated cells (right).

Fig 5

Caveolin-1 is important for infection by virions and ISVPs. 293 cells were transfected with empty vector or vector expressing dominant-negative caveolin-1. Cells were infected with T1L virions or ISVPs 24 h after transfection and then fixed and stained for μNS at 24 hpi. The number of transfected cells that were also μNS positive (infected) was determined by flow cytometry. The relative percentage of infected cells compared to empty vector (pEGFP-N1)-transfected controls is displayed. Error bars represent the standard errors of the means from three independent experiments.

Fig 6

Cholesterol plays a role in infection by reovirus virions. (A) L929 cells were pretreated with the indicated concentrations of MβCD, and viability was assessed at 24 hpi. A parallel set of samples was infected with T1L virions or ISVPs at an MOI of 3 and assayed for viral yield at 24 hpi. (B and C) L929 cells were pretreated with vehicle (water) or 5 mM MβCD and infected with reovirus T1L virions (B) or ISVPs (C) at an MOI of 3. Viral yields were determined at the indicated times postinfection by plaque assay on L929 cells. (D) L929 cells in suspension culture were pretreated with vehicle (water) or MβCD and adsorbed with reovirus virions (gray lines) or ISVPs (black lines) at a concentration of 1 × 10⁵ particles/cell or an equivalent volume of gel saline (shaded peaks). After adsorption, cells were fixed and stained with an anti-reovirus T1L antibody. Reovirus binding was quantified by flow cytometry. (E) Cells were treated with MβCD for 1 h, and 0.1 mM water-soluble cholesterol was added prior to or at the time of adsorption. Infection was analyzed by immunoblotting for μNS at 24 hpi. (F and G) A549 cells were pretreated with vehicle (water) or MβCD for 1 h. Treated cells were incubated with prelabeled transferrin (F) or CTB (G). Warm medium was added to the cultures, and after 15 min cells were fixed and ligand uptake was visualized by confocal microscopy. Scale bars represent 50 μM. (H) Reovirus virions (5 × 10¹⁰) were incubated with chymotrypsin, cholesterol, or both in virion dialysis buffer at 32°C. At the indicated times, digestions were terminated with PMSF and the particles were visualized by SDS-PAGE and Coomassie blue staining.