Mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of Eps15

Abstract

It has recently been shown that cell entry of mouse hepatitis virus type 2 (MHV-2) is mediated through endocytosis (Z. Qiu et al., J. Virol. 80:5768-5776, 2006). However, the molecular mechanism underlying MHV-2 entry is not known. Here we employed multiple chemical and molecular approaches to determine the molecular pathways for MHV-2 entry. Our results showed that MHV-2 gene expression and infectivity were significantly inhibited when cells were treated with chemical and physiologic blockers of the clathrin-mediated pathway, such as chlorpromazine and hypertonic sucrose medium. Furthermore, viral gene expression was significantly inhibited when cells were transfected with a small interfering RNA specific to the clathrin heavy chain. However, these treatments did not affect the infectivity and gene expression of MHV-A59, demonstrating the specificity of the inhibitions. In addition, overexpression of a dominant-negative mutant of caveolin 1 did not have any effect on MHV-2 infection, while it significantly blocked the caveolin-dependent uptake of cholera toxin subunit B. These results demonstrate that MHV-2 utilizes the clathrin- but not caveolin-mediated endocytic pathway for entry. Interestingly, when the cells transiently overexpressed a dominant-negative form (DIII) of Eps15, which is thought to be an essential component of the clathrin pathway, viral gene expression and infectivity were unaffected, although DIII expression blocked transferrin uptake and vesicular stomatitis virus infection, which are dependent on clathrin-mediated endocytosis. Thus, MHV-2 entry is mediated through clathrin-dependent but Eps15-independent endocytosis.

Figures

FIG. 1.

MHV-2 infection was impaired by inhibition of endosomal acidification. (A) DBT cells were pretreated with chloroquine (50 μM) (CH), bafilomycin A1 (100 nM) (BFA), or DMSO (Mock) for 0.5 h prior to infection. Cells were infected with MHV-2 (MHV2) or MHV-A59 (A59) at an MOI of 10. Chloroquine or bafilomycin A1 was present in the medium throughout the infection. At 7 h p.i., titers of virus were determined by plaque assay and were expressed as mean PFU/ml from one of the three triplicate experiments. Error bars indicate standard deviations of the means. (B to E) MHV-2 gene expression was inhibited by chloroquine. DBT cells were treated with chloroquine and infected with MHV-2 (B) or MHV-A59 (D) as described for panel A. At the indicated time points p.i., infected cells were collected and viral N protein was detected with Western blot analysis. β-Actin was used as an internal control for normalization. The amount of each protein band was quantified by densitometric analysis with UPV software. The amount of N protein was normalized to β-actin expressed in virus-infected cells and is presented as a relative amount in panels C and E for MHV-2 and MHV-A59, respectively. Error bars indicate standard deviations of the means. Data are representative of three independent experiments.

FIG. 2.

Suppression of MHV-2 propagation by treatment with chlorpromazine (CPZ). (A) DBT cells were pretreated with chlorpromazine (10 μg/ml) for 0.5 h, followed by infection with MHV-2 or MHV-A59 (A59) at an MOI of 10. Chlorpromazine was kept in the medium throughout the infection (+ CPZ). Mock-treated cells were used as a negative control (− CPZ). The virus titer was determined at 7 h p.i. by plaque assay and expressed in mean PFU/ml from one of the three triplicate experiments. Error bars indicate the standard deviations of the means. (B) Chlorpromazine-treated (+) or mock-treated (−) cells were infected with MHV-2 at an MOI of 10. Infected cells were collected at the indicated time points p.i., and the viral N protein was detected with Western blot analysis using β-actin as an internal control for normalization. (C) Quantification of protein bands shown in panel B. The amount of each protein band was quantified by densitometric analysis with UPV software. The amount of N protein was normalized to β-actin expressed in virus-infected cells and is presented as a relative amount. Error bars indicate standard deviations of the means. Data are representative of at least three independent experiments.

FIG. 3.

MHV-2 propagation was impaired by hypertonic sucrose treatment. (A and C) DBT cells were pretreated with hypertonic (400 mM) sucrose (+) medium or mock-treated (-) for 0.5 h, followed by infection with MHV-2 (A) or MHV-A59 (C) at an MOI of 10. Expression of viral N protein was detected at various time points p.i. by Western blot analysis, with β-actin as an internal control for normalization. (B and D) Quantification of the protein bands shown in panels A and C, respectively. The amount of the N protein was quantified by densitometric analysis with UPV software, normalized with β-actin expressed in virus-infected cells, and presented as a relative amount. All data are representative of three independent experiments.

FIG. 4.

Inhibition of MHV-2 infection by the actin cytoskeleton-modifying agent. (A and D) DBT cells were pretreated with nocodazole (N) or cytochalasin D (C) at 4 μg/ml or with DMSO as a mock control (M) for 0.5 h and then infected with MHV-2 (A) or MHV-A59 (D) at an MOI of 10. The drugs were kept in the medium throughout the infection. At the indicated time points p.i., viral N protein was determined by Western blotting, with β-actin as an internal control. (B, C, E, and F) DBT cells were treated with nocodazole (B and E) or cytochalasin D (C and F) either 0.5 h prior to infection (pretreatment-7 h p.i.) or 2 h after infection (2-7 h p.i.) at various concentrations (μg/ml) as indicated or treated with DMSO (Mock). DBT cells were infected with MHV-2 (B and C) or MHV-A59 (E and F) at an MOI of 10. Virus titers in the medium were determined at 7 h p.i. by plaque assay. The data are representative of three independent experiments and are indicated as mean PFU/ml from a triplicate experiment. Error bars indicate standard deviations of the means.

FIG. 5.

Inhibition of MHV-2 propagation by siRNA specific to clathrin heavy chain. (A) DBT cells were transfected with either siRNA specific to clathrin heavy chain (CH) or a negative, nonspecific control siRNA (NS). At 48 h posttransfection (p.t.), cells were lysed and the protein level for the endogenous clathrin heavy chain (HC) was detected by Western blotting with an antibody specific to the clathrin heavy chain. The level for β-actin detected was used as an internal control for normalization. (B) Quantification of the protein bands shown in panel A. The amounts of the clathrin heavy chain were quantified by densitometric analysis, normalized to those of β-actin, and expressed as relative amounts. (C and F) Following transfection with either CH siRNA or NS siRNA for 48 h as shown in panel A, cells were infected with MHV-2 (C) or MHV-A59 (E) at an MOI of 10. At various time points p.i., as indicated, cells were collected and the expression of viral N protein was detected by Western blot analysis with β-actin as an internal control for normalization. (D and F) Quantification of the viral N protein shown in panels C and E, respectively. The methods used for quantification were identical to those described for panel B. The differences in N protein expression between specific (CH) and nonspecific (NS) siRNA-transfected cells were statistically significant (P < 0.01) in MHV-2-infected cells (D) but were not significant (P > 0.05) in MHV-A59-infected cells. The data are representative of three independent experiments.

FIG. 6.

Overexpression of a dominant-negative mutant of caveolin 1 could not suppress MHV-2 infection. (A) Effect of dominant-negative caveolin 1 on MHV-2 infection. DBT cells were transfected with GFP-cav-1 (dn cav-1). At 24 h posttransfection, DBT cells were subjected to FACS to separate GFP-positive (+) and GFP-negative (−) cell populations, which were then separately infected with MHV-2 at an MOI of 10. Expression of the viral N protein was detected by Western blot analysis, with β-actin as an internal control. (B) Quantification of the protein bands shown in panel A. The amounts of the N protein were quantified by densitometric analysis, normalized to those of β-actin, and expressed as relative amounts. (C) Effect of dominant-negative caveolin 1 on cholera toxin subunit B (CT-B) uptake. DBT cells were transfected with GFP-cav-1 (dn cav-1) or cav-1-GFP (wt cav-1). At 24 h posttransfection, cells were incubated with Alexa Fluor 594-conjugated CT-B for 30 min, washed, and fixed in precooled (−80°C) methanol-acetone. The images were taken with an Olympus IX-70 microscope and a digital camera (MagnaFire). Note that detection of GFP in the cells indicates the expression of the caveolin 1 plasmids, while detection of conjugated CT-B at the cell surface indicates the blockage of internalization.

FIG. 7.

Overexpression of a dominant-negative mutant of Eps15 could not prevent MHV-2 infection. (A) Effect of dominant-negative Eps15 on MHV-2 infection. DBT cells were transfected with an Eps15 dominant-negative mutant, EGFP-DIII (DIII), or a control, EGFP-DIIIΔ2 (DIIIΔ2), which exhibits a similar effect on endocytosis to that of the wild-type Eps15, or mock transfected (MT). At 24 h posttransfection, cells were either infected with MHV-2 at an MOI of 10 or mock-infected (MI). At 6 h p.i., cells were fixed and stained for the viral N protein using an N-specific monoclonal antibody and PE conjugate. Flow cytometric analysis was used to quantify the expression of the EGFP (green) and viral N protein (red), which monitors the transfection (x axis) and infection (y axis), respectively. The percentage of cells within each quadrant is shown. Data were representative of three independent experiments. (B) Expression of dominant-negative Eps15 blocked transferrin uptake. DBT cells grown on glass coverslips were transiently transfected with either EGFP-DIII (DIII) or EGFP-DIIIΔ2 (DIIIΔ2) for 24 h. Alexa 594-labeled transferrin was bound to serum-starved cells for 20 min at 4°C. Cells were washed, transferred to 37°C for 15 min, washed with low-pH glycine to remove uninternalized ligand, and fixed. Cells were then observed under a dual-fluorescence microscope. The images were taken with an AxioCam MRc color camera using AvioVision software. White arrowheads in the upper right panel indicate that the EGFP-expressing cells (green) failed to take up transferrin (red), whereas the arrows in the lower right panel indicate the cells both expressing EGFP (green) and taking up transferrin (red). (C) Expression of dominant-negative Eps15 inhibited VSV gene expression. EGFP-DIII-transfected (DIII) or mock-transfected (MT) DBT cells were infected with VSV at an MOI of 10. Viral G protein expression was detected by Western blotting at various time points p.i., as indicated. β-Actin was used as an internal control. (D) Quantification of the protein bands shown in panel C. The amount of the G protein was quantified by densitometric analysis with UPV software, normalized with β-actin expressed in virus-infected cells, and presented as the mean relative amount. Error bars indicate the standard deviations of the means. Data are representative of three independent experiments.

FIG. 8.

MHV-2 gene expression and propagation were unaffected by the expression of the dominant-negative mutant of Eps15. DBT cells were transfected with the dominant-negative mutant of Eps15, EGFP-DIII. At 24 h posttransfection, DBT cells were subjected to FACS to separate EGFP-positive (+) and EGFP-negative (−) cell populations, which were then separately infected with MHV-2 at an MOI of 10. (A) Expression of the viral N protein was detected by Western blot analysis, with β-actin as an internal control. (B) Quantification of the protein bands shown in panel A. The amounts of the N protein were quantified by densitometric analysis and normalized with β-actin. (C) Titers of virus were determined at the indicated time points p.i. by plaque assay in triplicate and expressed as mean PFU/ml. Error bars indicate the standard deviations of the means. All data are representative of at least three independent experiments.