SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells

Abstract

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.

FIGURE 1

SARS-CoV S-protein-mediated pseudovirus entry is blocked by cathepsin L inhibitor. HEK293T cells were transfected with plasmids encoding human ACE2 or the HIV-1 receptors CD4 and CXCR4, replated, and infected with GFP-expressing MLV virus pseudotyped with the SARS-CoV S protein or HIV-1 envelope glycoprotein (SARS/MLV or HIV-1/MLV, respectively). Target cells were preincubated for 3 h with indicated concentrations of protease inhibitors (A) or cathepsin inhibitors (B) and incubated for 5 h with the indicated pseudoviruses and inhibitors. Infected cells were trypsinized 48–60 h later, and GFP expression was measured by flow cytometry. C, fluorescent micrographs of cells infected in the presence and absence of 10 μm cathepsin L inhibitor. Abbreviations used are: Phsdn, phosphoramidon; Pep A, pepstatin A; Cath L, cathepsin L.

FIGURE 2

Cathepsin (Cath) L inhibitor suppresses infectious SARS-CoV. Vero 118 cells (SARS-CoV, HCoV-NL63) and LLC-MK2 cells (HCoV-NL63) were infected with replication-competent SARS-CoV or HCoV-NL63 in the presence of indicated protease inhibitors (A) or cathepsin inhibitors (B) as described under”Experimental Procedures.“Sindbis virus and VSV are included as controls in both cell lines. Cells were fixed 8 h (for experiments with SARS-CoV, Sindbis virus, and VSV) or 24 h (for those with HCoV-NL63) post-infection, permeabilized and stained with ferret sera against SARS-CoV or with human serum against HCoV-NL63, as described. Positively stained cells were counted. Phsdn, phosphoramidon.

FIGURE 3

HCoV-NL63 S-protein-mediated pseudovirus entry is not affected by cathepsin (Cath) inhibitors. Human ACE2-transfected HEK293T cells were preincubated for 3 h (A, B) or 30 min (C) with infection inhibitors and then incubated with SARS/MLV, NL63/MLV, HIV/MLV, or VSV-G/MLV in the presence of indicated cathepsin inhibitors (A), the broad cysteine protease inhibitor E64d (B), or NH₄Cl, an inhibitor of endosomal acidification (C). Infectivity was assessed as described under”Experimental Procedures.“

FIGURE 4

Exogenous cathepsin (Cath) L enhances the entry of SARS/MLV, but not that of NL63/MLV.A, HEK293T cells were transfected with cathepsin B, L, or S expressor plasmids and various amounts of human ACE2 plasmid as described under”Experimental Procedures.“Cells were divided and replated for infection, metabolic labeling, or flow cytometry the following day. Two days post-transfection cells were incubated with SARS/MLV, NL63/MLV, or VSV-G/MLV. In parallel, cell surface ACE2 expression was assessed by flow cytometry with anti-human-ACE2 antibody. Infection by GFP-expressing pseudovirions, measured 2 days post-infection by flow cytometry (vertical axis), is plotted here against ACE2 expression (horizontal axis). B, approximately 36 h post-transfection, an aliquot of transfected cells used in A was labeled overnight with [³⁵S]cysteine and [³⁵S]methionine. Cells were lysed in 0.5% Nonidet P-40/PBS containing protease inhibitor mixture, immunoprecipitated with 1D4 antibody recognizing a C-terminal epitope present on the cathepsin protein, and analyzed by SDS-PAGE. Three lanes each for cathepsins B, L, and S represent three transfections (in the order of increasing amounts of ACE2 DNA). C, MEFs cultured from a cathepsin L^–/– mouse were transduced with VSV-G/MLV carrying the human ACE2 gene and, 5 h later, with VSV-G/MLV carrying the human cathepsin L gene. Cells were split and infected with SARS/MLV, NL63/MLV, MARV/MLV, Ebola/MLV, or VSV-G/MLV. Infection efficiency was assessed 3 days later by measuring GFP expression by flow cytometry and is expressed as a percentage of GFP-expression observed with cells transfected with ACE2 alone.