Endocytosis of murine norovirus 1 into murine macrophages is dependent on dynamin II and cholesterol - PubMed (original) (raw)

Endocytosis of murine norovirus 1 into murine macrophages is dependent on dynamin II and cholesterol

Jeffrey W Perry et al. J Virol. 2010 Jun.

Abstract

Although noroviruses cause the vast majority of nonbacterial gastroenteritis in humans, little is known about their life cycle, including viral entry. Murine norovirus (MNV) is the only norovirus to date that efficiently infects cells in culture. To elucidate the productive route of infection for MNV-1 into murine macrophages, we used a neutral red (NR) infectious center assay and pharmacological inhibitors in combination with dominant-negative (DN) and small interfering RNA (siRNA) constructs to show that clathrin- and caveolin-mediated endocytosis did not play a role in entry. In addition, we showed that phagocytosis or macropinocytosis, flotillin-1, and GRAF1 are not required for the major route of MNV-1 uptake. However, MNV-1 genome release occurred within 1 h, and endocytosis was significantly inhibited by the cholesterol-sequestering drugs nystatin and methyl-beta-cyclodextrin, the dynamin-specific inhibitor dynasore, and the dominant-negative dynamin II mutant K44A. Therefore, we conclude that the productive route of MNV-1 entry into murine macrophages is rapid and requires host cholesterol and dynamin II.

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Figures

FIG. 1.

Uptake of fluorescently labeled transferrin is inhibited by a hypotonic solution of sucrose or chlorpromazine. RAW 264.7 cells were incubated with vehicle control (A, D, and G), chlorpromazine (B), a hypotonic solution of sucrose (C), or nystatin (F) for 30 min or MβCD (E and H) or MβCD and 10% FBS (I) for 60 min before incubation with 50 μg/ml fluorescently labeled transferrin (A to E) or 10 μg/ml fluorescently labeled cholera toxin subunit B (F to I). Cells were washed and media containing inhibitor or vehicle control added back for 5 min for transferrin or 60 min for cholera toxin subunit B. Cells were fixed in 4% paraformaldehyde and mounted with Prolong Gold Antifade with DAPI (Invitrogen, CA). Cells were examined using an Olympus IX70 inverted microscope and images acquired using the Metamorph Premier version 6.3 image analysis software (Molecular Devices, Downington, PA).

FIG. 2.

Neutral red (NR) infectious center assay confirms a pH-independent and sialic acid-dependent entry mechanism for MNV-1. (A, C, and D) Flow charts of the NR assay describing different treatment conditions. (A and B) MNV-1 rapidly becomes insensitive to light exposure. RAW 264.7 cells were infected with NR-containing virus at an MOI of 0.001, rocked at room temperature, exposed to light at 0, 15, 30, 45, 60, and 75 min postinfection (pi), and overlaid with agarose, and plaques were counted 48 to 72 h pi. (C, D, and E) MNV-1 entry is sialic acid dependent and pH independent. (C and E, pretreatment) RAW 264.7 cells were pretreated with 2.5 mU/ml Vibrio cholerae neuraminidase (Neura) or 200 μM chloroquine (Chloro) for 30 min, infected by rocking for 60 min, and overlaid with media containing agarose, and plaques were counted 48 to 72 h pi. (D and E, posttreatment) Alternatively, RAW 264.7 cells were infected with an MOI of 0.001 for 60 min and then posttreated with 2.5 mU/ml Vibrio c holerae neuraminidase (Neura) or 200 μM chloroquine (Chloro) for a total of 90 min before performing the NR assay. (E, posttransfer) In case of Vibrio cholerae neuraminidase treatment, RAW 264.7 cells were posttreated as described, scraped, and transferred to an untreated monolayer before a plaque assay was performed and viral titers were determined. *, P < 0.05.

FIG. 3.

MNV-1 infection requires dynamin II. RAW 264.7 cells (A) and BMDMs (B) were pretreated with dynasore at the indicated concentration for 30 min, infected with MNV-1 or VSV on ice for 1 h, and washed with PBS. At 8 h (RAW 264.7 cells) or 10 h (BMDMs) postinfection, cells were freeze-thawed two times and viral titers determined by plaque assay. (C) RAW 264.7 cells were transfected with a wt and DN construct of GFP-tagged dynamin II and then infected with MNV-1 (MOI of 10) for 12 h. The number of VPg-expressing cells was determined by immunofluorescence and normalized to the wt control. (D) RAW 264.7 cells were infected with NR-containing virus at an MOI of 0.001, with rocking at room temperature for 60 min. RAW 264.7 cells were either pretreated (pretreatment) or treated for 60 min postinfection (posttreatment) with 80 μM dynasore before performing a plaque assay and determining viral titers. *, P < 0.05; ***, P < 0.001.

FIG. 4.

MNV-1 infection is clathrin independent. (A) RAW 264.7 cells were infected with NR-containing virus at an MOI of 0.001 and rocked at room temperature for 60 min. RAW 264.7 cells were either pretreated (pretreatment) or treated for 60 min pi (posttreatment) with 40 μM chlorpromazine (Chloro) or 300 mM sucrose. (B) RAW 264.7 cells were transfected with a GFP-only (GFP), GFP-tagged wt (EPS 15 wt), or DN construct (EPS 15 DN) of EPS 15 and then infected with MNV-1 at an MOI of 10 for 12 h. The number of VPg-expressing cells was determined by immunofluorescence and normalized to the value for the GFP-only control. (C) RAW 264.7 cells were incubated with an Accell siRNA clathrin heavy-chain construct (CHC siRNA) or a nontargeting construct (NT siRNA) (Dharmacon) for 72 h. Cells were then infected with MNV-1 or VSV at an MOI of 10. The number of VPg-expressing cells was determined by immunofluorescence 12 h after infection and normalized to the value for the NT control. (D) To verify clathrin heavy-chain protein knockdown, protein samples from cells expressing each siRNA construct were analyzed by immunoblotting for clathrin heavy chain, and protein levels were quantitated as described in the text. *, P < 0.05; ***, P < 0.001.

FIG. 5.

MNV-1 infection is caveolin independent. (A) BMDMs were isolated from caveolin 1 knockout mice and wt controls and infected with MNV-1 at an MOI of 2. Viral titers were determined at times indicated. (B) RAW 264.7 cells were transfected with a DN GFP-tagged caveolin 1 (CAV 1 DN) and a GFP-only control (GFP) construct and infected with MNV-1 at an MOI of 10 for 12 h. VPg-expressing cells were determined by immunofluorescence and normalized to the value for the GFP-only control. (C) RAW 264.7 cells were incubated with an Accell siRNA caveolin 1 construct (CAV 1 siRNA) or a nontargeting construct (NT siRNA) (Dharmacon) for 72 h. Cells were then infected with MNV-1 or VSV at an MOI of 10, and VPg expression was determined by immunofluorescence 12 h after infection. The number of VPg-expressing cells was normalized to the value for the NT control. (D) To verify caveolin 1 protein knockdown, protein samples from cells expressing each siRNA construct were analyzed by immunoblotting for caveolin 1 and quantitated as described in the text.

FIG. 6.

MNV-1 infection is independent of phagocytosis and/or macropinocytosis. (A) RAW 264.7 cells were pretreated with 10 μM cytochalasin D (Cyto D), 200 μM amiloride (EIPA), or mock control before infection with Listeria monocytogenes strain 10403S at an MOI of 1 for 30 min at 37°C. Replication of noninternalized bacteria was inhibited with 50 μg/ml gentamicin. After 1 h postinfection, cells were lysed and internalized bacteria plated onto LB plates. A total of 24 h after incubation at 37°C, CFU were quantitated and normalized to the value for the mock control. (B) RAW 264.7 cells were infected with NR-containing virus at an MOI of 0.001 and rocked at room temperature for 60 min. RAW 264.7 cells were either pretreated (pretreatment) or treated for 60 min pi (posttreatment) with 10 μM cytochalasin D (cyto D), 200 μM amiloride (EIPA), or mock control. (C) RAW 264.7 cells were transfected with a wt (RAC 1 wt) and a DN (RAC 1 DN) GFP-tagged Rac 1 and infected with MNV-1 at an MOI of 10 for 12 h. The number of VPg-expressing cells was determined by immunofluorescence and normalized to the value for the wt control. *, P < 0.05; ***, P < 0.001.

FIG. 7.

MNV-1 infection is cholesterol dependent. (A) RAW 264.7 cells were infected with NR-containing virus at an MOI of 0.001 and rocked at room temperature for 60 min. RAW 264.7 cells were either pretreated (pretreatment) or treated for 60 min pi (posttreatment) with 50 μM nystatin, 2 mM MβCD, or 2 mM MβCD with 10% fetal bovine serum (MβCD & FBS). (B) RAW 264.7 cells or BMDMs were infected with MNV-1 for 12 h after pretreatment with 2 mM MβCD or 2 mM MβCD with 10% fetal bovine serum (MβCD & FBS). The number of VPg-expressing cells was determined by immunofluorescence and normalized to the value for a no-treatment control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 8.

The major infectious route for MNV-1 is independent of flotillin-1 and GRAF1. RAW 264.7 cells were incubated with an Accell siRNA against flotillin-1 (A), GRAF1 (C), or a nontargeting (NT) construct (A and C) (Dharmacon) for 72 h. Cells were then infected with MNV-1 at an MOI of 10, and VPg expression was determined by immunofluorescence 12 h after infection. To verify flotillin-1 (FLOT) (B) or GRAF1 (D) protein knockdown, protein samples of cells transfected with the respective siRNA construct were analyzed by immunoblotting for flotillin-1 or GRAF1 and quantitated as described in the text.

FIG. 9.

Clathrin- and caveolin-dependent endocytosis or phagocytosis/macropinocytosis are not a minor route of entry for MNV-1. RAW 264.7 cells were pretreated with 2 mM MβCD alone (−), 2 mM MβCD and 10 μM cytochalasin D (cyto D), 2 mM MβCD and 200 μM amiloride (EIPA), 2 mM MβCD and 80 μM dynasore (Dyna), or 2 mM MβCD and 40 μM chlorpromazine (Chloro). RAW 264.7 cells were infected with NR-containing virus at an MOI of 0.001 and rocked at room temperature for 60 min before performing the NR assay and determining viral titers. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Endocytosis of murine norovirus 1 into murine macrophages is dependent on dynamin II and cholesterol - PubMed (original) (raw)