Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry

Abstract

Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.

FIG. 1.

Effect of ammonium chloride (NH₄Cl, a weak base) (A) and bafilomycin A1 (BAF, a vacuolar H⁺/ATPase inhibitor) (B) on viral replication in L2 cells. Cells were either pretreated from 1 h before virus infection to 1 h postinfection (p.i.) or treated from 1 to 3 h p.i. with 40 mM NH₄Cl or 100 nM BAF. After treatment, medium containing the agents was removed from cells and replaced with fresh growth medium. Culture supernatants were collected at 16 h p.i., and viral titers were determined by plaque assay. Error bars represent the standard deviations from three replicates.

FIG. 2.

Schematic diagram of chimeric isogenic A59 recombinant viruses expressing A59 (white) or MHV-2 (gray) spike genes. A59 spike is cleaved by furin-like enzymes at a site with dibasic amino acids (RRAHR) into S1 and S2 subunits. However, with one less basic residue, the proteolytic cleavage site of MHV-2 is not functional (HRARS) and the spike is not cleaved. Using targeted RNA recombination, chimeric A59 recombinant viruses expressing MHV-2 spike and MHV-2 spike with a S757R substitution (HRARR) were selected, termed RA59/MHV-2S and RA59/MHV-2S_S757R, respectively. The relevant substitution is underlined. The MHV-2 genome shown in gray is not a recombinant. Genomes are not shown to scale.

FIG. 3.

(A and B) Pretreatment with NH₄Cl diminishes RA59/MHV-2S_EGFP infection. L2 cell monolayers were pretreated from 1 h before virus infection to 1 h postinfection with 40 mM NH₄Cl. After treatment, medium containing NH₄Cl was removed from cells and replaced with fresh growth medium. Images were recorded at 8 h postinfection. (C and D) Trypsin, but not low-pH treatment, overcomes the NH₄Cl block. After viral adsorption in the presence of NH₄Cl, cells were incubated with PBS (pH 7.4) at 37°C for 15 min and then treated either with pH 5.0 buffer for 15 min (C) or with 10 μg/ml trypsin for 5 min (D) and then washed thoroughly with PBS. NH₄Cl was present during the entire process and for another 1 h after low-pH or trypsin treatment. Images were recorded after subsequent 7-h incubation in fresh medium without NH₄Cl.

FIG. 4.

(A) Trypsin, but not low-pH treatment, induces MHV-2 spike-mediated cell-cell fusion. L2 cells were infected with RA59/MHV-2S. At 24 h postinfection, cells were washed with PBS and incubated for 5 min with PBS at pH 5.0 or PBS at pH 7.4 containing 0, 5, or 20 μg/ml trypsin. PBS was then replaced with fresh medium, and images were recorded after 1 h of incubation. The bar represents 50 μm. (B) Immunoblot of MHV spike proteins in infected cell lysates following trypsin or low-pH treatment as described in the legend to panel A.

FIG. 5.

Effect of endosomal cysteine protease inhibitors on infectivity of RA59_EGFP and RA59/MHV-2S_EGFP in L2 cells. Cells were pretreated with various concentrations of protease inhibitors E-64 (cysteine protease inhibitor) (A), CA-074 (specific cathepsin B inhibitor) (B), and Z-FY-DMK (cathepsin L/B inhibitor) (C) for 3 h before adding serially diluted EGFP-expressing viruses. After 1 h of adsorption, the viral inocula were removed and the cells were incubated with medium containing the same drug for another hour. The cells were then incubated with fresh growth medium without drugs, and infectivity was measured at 8 h postinfection by counting EGFP-positive cells. Infectivity values are reported as infectivity units (iu)/ml virus, where one infectivity unit corresponds to one EGFP-positive cell. Infectivity of EGFP-expressing viruses in the presence of inhibitors was expressed as a percentage of infectivity observed in mock-treated cells. Error bars represent the standard deviations from at least three replicates.

FIG. 6.

(A) Effect of S757R substitution in MHV-2 spike on cell-cell fusion. L2 cells were infected with isogenic A59 recombinant viruses expressing spike of MHV-2, MHV-2_S757R, and A59 as labeled in the figure. Images were recorded at 9 h and 24 h postinfection. The bar represents 50 μm. (B) Immunoblot of MHV spike proteins in lysates of cells infected with the viruses described for panel A. In addition to the uncleaved and cleaved spike bands, an extra spike-related band of about 120 kDa, which has been previously observed (13, 14), was detected in lysates of cells infected with A59.

FIG. 7.

Effect of NH₄Cl on RA59/MHV-2S_S757R replication in L2 cells. Cells were either pretreated from 1 h before virus infection to 1 h p.i. or treated from 1 to 3 h p.i. with 40 mM NH₄Cl. After treatment, medium containing NH₄Cl was removed from cells and replaced with fresh growth medium. Culture supernatants were collected at 16 h p.i., and viral titers were determined by plaque assay. Error bars represent the standard deviations from three replicates.

FIG. 8.

Effect of endosomal cysteine protease inhibitors on RA59/MHV-2S (A) and RA59/MHV-2S_S757R (B) replication in L2 cells. Cells were pretreated with 100 μM E-64 (to inactivate endosomal cysteine proteases) or 10 μM Z-FY-DMK (to specifically inactivate cathepsin B and L) for 3 h, and then infected with viruses in the presence of inhibitor for 1 h. Growth medium containing inhibitors was then removed from cells and replaced with fresh medium without inhibitors. At indicated times (10, 14, and 18 h), supernatants were harvested and viral yields were determined by plaque assay.