Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II

Abstract

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.

Figure 1

Chemical inhibitors against clathrin-, dynamin-, and cholesterol-mediated endocytosis reduce PSaV infection. A–C Confluent monolayers of LLC-PK, Caco-2, and MA104 cells were treated with DMSO vehicle or different concentrations of chlorpromazine (CPZ), MβCD, nystatin, or dynasore prior to infection with PSaV Cowden (A), Coxsackievirus B3 (CVB3) Nancy (B), or human rotavirus Wa (C) strains. Soluble cholesterol was added to the medium to examine the effect of cholesterol replenishment following MβCD-mediated depletion and then cells were infected with PSaV Cowden, CVB3 Nancy, or human rotavirus Wa strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the result obtained with control DMSO-treated cells. Data for panels A–C are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. *P < 0.05; **P < 0.001; ***P < 0.0001.

Figure 2

Small interfering RNAs (siRNAs) against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. A LLC-PK cells transfected with scrambled siRNA (Scram) or siRNAs against clathrin heavy chain (CHC), dynamin II, or caveolin-1 were harvested at 24 and 48 h post-transfection. The down-regulation of each protein by siRNA knock-down was evaluated by western blotting analysis using antibodies specific for each protein. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane. The cells transfected with each siRNA and then incubated were infected with PSaV Cowden (B), CVB3 Nancy (C), or rotavirus Wa (D) strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the results obtained in the scrambled siRNA-transfected cells. Data for panels B–D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. *P < 0.05; **P < 0.001; ***P < 0.0001.

Figure 3

Dominant negative (DN) mutants against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. The cells transfected with GFP-tagged wild type (WT) or dominant negative (DN) Eps15, caveolin-1, or dynamin II were incubated with PSaV Cowden (A), CVB3 Nancy (B), or rotavirus Wa (C) strains. Infected GFP-expressing cells were counted after being stained with antibodies specific for each virus. Data for panels A–C are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. *P < 0.05; **P < 0.001; ***P < 0.0001.

Figure 4

PSaV entry and RNA release depend on clathrin-, dynamin-, and cholesterol-mediated endocytosis. A LLC-PK cells grown in 6-well plates were incubated with neutral red (NR)-labeled PSaV Cowden strain for the indicated times and then exposed or not to UV light. The supernatants of each cell lysate were inoculated into fresh LLC-PK monolayers in 6-well plates, overlaid with agar, and incubated for 4 days. Results are shown as percentages to the number of plaques in the unilluminated control. The NR infectious center assay was performed in cells pretreated with DMSO vehicle or 20 μM chlorpromazine (CPZ), or transfected with scrambled siRNA or CHC siRNA (B); pretreated with DMSO vehicle or 100 μM dynasore (Dyna), or transfected with scrambled siRNA or dynamin II (Dyn II) siRNA (C); or pretreated with DMSO vehicle, 20 mM MβCD for 1 h, or MβCD followed by incubation with 200 μM soluble cholesterol for 30 min (D). The cells were then incubated with NR-labeled PSaV for 120 min. The number of plaques was normalized to those obtained with unilluminated controls exposed to the above conditions. Data for panels A–D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. *P < 0.05; **P < 0.001; ***P < 0.0001.

Figure 5

PSaV entry depends on Rab5 and Rab7. A, B LLC-PK cells were transfected with scrambled siRNA or siRNAs against Rab5 (A) or Rab7 (B). Next, cells were exposed to AF594-labeled PSaV particles (approximately 415 particles per cell) for EEA1 colocalization or for LAMP2 colocalization. This experiment was done in triplicate and representative images are shown. The scale bars correspond to 10 μm. The bottom portion displays immunoblots to confirm silencing levels of Rab5 and Rab7 by transfection of each corresponding siRNA. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane.

Figure 6

PSaV infection depends on Rab5 and Rab7. A–D LLC-PK, Caco-2, and MA104 cells were transfected with scrambled siRNA or siRNAs against Rab5 or Rab7, and then incubated with PSaV Cowden (A), CVB3 Nancy (B), or rotavirus Wa (C) strains, respectively. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, normalized to the results obtained in the scrambled siRNA-transfected cells. D The virus titer was determined by TCID₅₀ using the cell lysates harvested from the cells in the above experimental conditions. Data for panels A–D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using the one-way ANOVA. *P < 0.05; **P < 0.001; ***P < 0.0001. E The level of PSaV VPg protein production in the above conditions was determined by western blotting analysis. GAPDH was used as a loading control. The intensity of PSaV VPg protein relative to GAPDH was determined by densitometric analysis and is indicated above its lane.

Figure 7

PSaV entry is pH-dependent and involves actin and microtubules. A, B Mock- or chloroquine-treated LLC-PK cells were inoculated with the PSaV Cowden strain and then incubated with CMFDA to visualize acidification of intracellular compartments, and incubated for further 30 min in maintenance media (A) or incubated with CMFDA followed by LAMP2 antibody to check colocalization by confocal microscopy (B). C, D After pretreatment of LLC-PK cells with chloroquine and infection with AF594-labeled PSaV particles (approximately 415 particles per cell), cells were incubated with neutral (pH 7.2) (C) or acidic (pH 5.2) buffers (D). The cells were prepared for confocal microscopy to check colocalization of AF594-labeled PSaV particles with LAMP2. All experiments were performed in triplicate and panels A–D show representative sets of results. Scale bars for panels A–D correspond to 10 μm.