Two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis

Abstract

The coronaviruses mouse hepatitis virus type 2 (MHV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV) utilize proteases to enter host cells. Upon receptor binding, the spike (S) proteins of both viruses are activated for membrane fusion by proteases, such as trypsin, present in the environment, facilitating virus entry from the cell surface. In contrast, in the absence of extracellular proteases, these viruses can enter cells via an endosomal pathway and utilize endosomal cathepsins for S protein activation. We demonstrate that the MHV-2 S protein uses multistep conformational changes for membrane fusion. After interaction with a soluble form of the MHV receptor (CEACAM1a), the metastable form of S protein is converted to a stable trimer, as revealed by mildly denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liposome-binding assays indicate that the receptor-bound virions are associated with the target membrane through hydrophobic interactions. The exposure of receptor-bound S protein to trypsin or cathepsin L (CPL) induces the formation of six-helix bundles (6HB), the final conformation. This trypsin- or CPL-mediated conversion to 6HB can be blocked by a heptad repeat peptide known to block the formation of 6HB. Although trypsin treatment enabled receptor-bound MHV-2 to enter from the cell surface, CPL failed to do so. Interestingly, consecutive treatment with CPL and then chlorpromazine enabled a portion of the virus to enter from cell surface. These results suggest that trypsin suffices for the induction of membrane fusion of receptor-primed S protein, but an additional unidentified cellular factor is required to trigger membrane fusion by CPL.

FIG. 1.

Schematic structure and step 1 conformational change of MHV-2 S protein induced by receptor binding. (A) The S protein has an N-terminal signal peptide (SP), a receptor-binding domain (RBD), a fusion peptide (FP), two heptad repeats (HR-N and HR-C), a transmembrane domain (TMD), and a cytoplasmic tail (CT). The putative cleavage site in the central region is identified by a white arrow. Epitopes recognized by the two antibodies (MAb 10G and anti-S2A) are depicted. (B) MHV-2 virions were incubated with various concentrations of soMHVR at 37°C for 30 min. The samples were boiled (lanes 1, 2, 8, and 10) or left unboiled (lanes 3 to 7 and lanes 10 to 14) and analyzed by Western blotting with the indicated antibody. (C) Precleaved and uncleaved S proteins of MHV-JHM-srr7 and MHV-2 were incubated in the presence or absence of soMHVR to compare the trimer formation. The samples were analyzed by native PAGE and Western blotting with the indicated antibody.

FIG. 2.

Hydrophobic interactions of receptor-primed MHV-2 with liposome. Virions incubated in the absence of liposomes and soMHVR (□) or in the presence of liposomes only (░⃞) or both liposomes and soMHVR (▪) were overlaid onto the top fraction of a step gradient and spun at 40,000 rpm for 1 h. The top, middle, and bottom fractions were drawn from the air-fluid interface, and viral RNAs were isolated from the fractions. Viral genomic RNA was quantified by real-time RT-PCR. The vertical line extending to the right of the bar indicates the standard deviation of four different experiments.

FIG. 3.

Step 2 conformational change of MHV-2 S protein is induced by trypsin. (A) soMHVR concentration-dependent conformational changes induced by trypsin. Virions were incubated with various concentrations of soMHVR (lanes 3 to 6) or PBS (lanes 1 and 2), and then the samples were digested with trypsin (lanes 2 to 6). (B) Comparison of the trypsin-digested S fragment between MHV-2 and MHV-JHM-srr7. Samples were treated with 1 μM soMHVR and 1 μg of trypsin/ml as in panel A. (C) Analysis of conformational changes induced by trypsin by proteinase K digestion. Virions were incubated in the absence (lanes 2, 4, 6, 8, 10, 12, 14, and 16) or presence (lanes 1, 3, 5, 7, 9, 11, 13 and 15) of soMHVR. The samples were digested with trypsin (lanes 3, 4, 7, 8, 11, 12, 15, and 16) and then further digested with proteinase K (lanes 5 to 8 and lanes 13 to 16) and analyzed by SDS-PAGE and Western blotting with the indicated antibody.

FIG. 4.

Step 2 conformational change of MHV-2 S protein is induced by cathepsins. (A) Analysis of conformational changes induced by CPL or CPB. Virions incubated with soMHVR were digested with CPL (lanes 5, 6, 7, and 8), CPB (lanes 9, 10, 11, and 12), or CPL plus CPB (lanes 13, 14, 15, and 16) and digested with proteinase K (lanes 3, 4, 7, 8, 11, 12, 15, and 16). (B) Comparison of cleaved fragments between trypsin and CPL treated S protein after interaction with soMHVR. Samples digested with trypsin or CPL as shown in Fig. 3C, lane 4, and Fig. 4A, lane 6, were loaded in lanes 1 and 3, respectively. A mixture of trypsin and CPL treated S protein was loaded in lane 2.

FIG. 5.

S Inhibition of conformational changes by HRP. All samples were treated with soMHVR and then mixed with HRP (lanes 2, 4, 6, 8, 10, and 12) or PBS (lanes 1, 3, 5, 7, 9, and 11). The samples were treated with trypsin (lanes 3 to 6) or CPL (lanes 9 to 12) and then with proteinase K (lanes 5, 6, 11, and 12) or PBS (lanes 1 to 4 and lanes 7 to 10).

FIG. 6.

Entry of MHV-2 from the cell surface is facilitated by protease and CPZ. (A) Effect of proteases on MHV-2 entry into DBT cells treated with bafilomycin. DBT cells were treated with bafilomycin for 30 min and infected with MHV-2 at a multiplicity of infection of 1. The cells were treated with trypsin or CPL and cultured in the presence of bafilomycin for a further 5 h. The amount of viral mRNA7 was measured quantitatively by real-time PCR. Cells not treated with bafilomycin or cells treated with bafilomycin but not with protease were used as controls. (B) Effect of CPZ on MHV-2 entry after treatment with CPL. DBT cells were treated with bafilomycin, infected with MHV-2, and treated with CPL as described for panel A. Immediately after CPL treatment, various concentrations of CPZ (pH 5.0) were treated for 3 min, and cells were cultured in the presence of bafilomycin for a further 5 h. The amount of viral mRNA7 was measured as described for panel A. Cells not treated with CPL or cells treated with CPZ but not with CPL were used as controls. baf, bafilomycin.

FIG. 7.

Model of the two-step conformational changes of MHV-2 S protein. After receptor interaction at the cell surface, MHV-2 S protein forms a stable trimer from its metastable state, and the fusion peptide is exposed and inserted into the target membrane (step 1). Upon treatment with trypsin, the receptor-primed form of MHV-2 S protein is digested to a 65-kDa fragment, which forms a stable 6HB to make a fusion pore (step 2). However, upon treatment with CPL, S protein cannot make a fusion pore even when 6HB formation is induced. CPZ treatment facilitates pore formation of CPL-primed S protein, allowing delivery of the viral core into the cytoplasm.