Retroviruses Pseudotyped with the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Efficiently Infect Cells Expressing Angiotensin-Converting Enzyme 2

Abstract

Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2).Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-proteinpseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.A distinct coronavirus (SARS-CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS), an acute pulmonary syndrome characterized by an atypical pneumonia that results in progressive respiratory failure and death in close to 10% of infected individuals (8,11,14,15). SARS-CoV is not closely related to any of the three previously defined genetic and serological coronavirus groups, although it may be distantly related to group 2 coronaviruses (21); the SARS-CoV spike (S) protein, a surface glycoprotein that mediates coronavirus entry into receptor-bearing cells, is also distinct from those of other coronaviruses (18,20). Reflecting this difference, SARS-CoV does not utilize any previously identified coronavirus receptors to infect cells. Rather, as our group have recently demonstrated, angiotensin-converting enzyme 2 (ACE2) serves as a functional receptor for this coronavirus (16,24,25).A quantitative system utilizing a well-characterized retroviral vector (1) for measuring SARS-CoV S-protein-mediated infection would obviate the need for specialized biosafety facilities for many studies, including those assessing humoral responses to potential vaccines. Here we show that simian immunodeficiency virus (SIV) pseudotyped with several codon-optimized S-protein variants could efficiently infect Vero E6 cells and HEK293T cells transiently or stably expressing ACE2. One such variant, truncated at its cytoplasmic tail and bearing instead a region of the tail of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (17), was especially efficient at mediating infection. Murine leukemia virus (MLV) pseudotyped with this S-protein variant also infected ACE2-ex...

SARS Coronavirus, but Not Human Coronavirus NL63, Utilizes Cathepsin L to Infect ACE2-expressing Cells

“…SARS-CoV infection is sensitive to NH 4 Cl, an inhibitor of lysosomal acidification (45,46), and roles for the lysosomal cysteine protease cathepsins in reovirus and ebolavirus infection have been described (32)(33)(34)(35)(36)41). Accordingly, we investigated the ability of cathepsin inhibitors to block infection mediated by the SARS-CoV S protein.…”

“…Inhibitors for cathepsin B (CA-074 methyl ester) and B/L (Z-LLL-FMK) were purchased from Sigma. Pseudotyped Virus Production and Infection-MLV-GFP virions pseudotyped with SARS-CoV S protein have been described previously (45). The experiments described here utilize S proteins of SARS-CoV and HCoV-NL63 with 27-and 24-residue deletions, respectively, in their cytoplasmic C termini.…”

“…The experiments described here utilize S proteins of SARS-CoV and HCoV-NL63 with 27-and 24-residue deletions, respectively, in their cytoplasmic C termini. We have previously shown that such deletions enhance incorporation of the S proteins into the retroviral particle (45). Plasmids encoding the SARS-CoV or HCoV-NL63 S proteins were cotranfected with plasmid encoding the MLV gag and pol genes and with a construct bearing the MLV long terminal repeats, packaging signal, and the green fluorescent protein (GFP) gene (pQ-GFP), derived from the pQCXIX vector (Invitrogen).…”

Recombinant Ov-ASP-1, a Th1-Biased Protein Adjuvant Derived from the Helminth Onchocerca volvulus, Can Directly Bind and Activate Antigen-Presenting Cells

“…As responder cells in the MLR, naive CD4 ϩ T cells were isolated by negative selection from five allogeneic normal healthy donors using a CD4 ϩ T cell Isolation Kit II (Miltenyi Biotec), according to the manufacturer's recommended protocol. Purified (23,24). The cells were maintained in DMEM (Invitrogen) supplemented with 10% FCS, 100 U/ml penicillin (PAA), 0.1 g/ml streptomycin (PAA), and 200 mM L-glutamine (Invitrogen) in a 5% saturated CO 2 atmosphere at 37°C.…”

“…A SARS-CoV pseudovirus system was developed in our laboratory as previously described (23,24). In brief, human embryonic kidney cells (HEK) 293 T cells were cotransfected with a plasmid encoding the S protein corresponding to SARS-CoV Tor2 isolate and a plasmid encoding Envdefective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) by using FuGENE 6 reagents (Boehringer Mannheim).…”