Chloroquine is a potent inhibitor of SARS coronavirus infection and spread
Severe acute respiratory syndrome (SARS) is caused by a newly discovered coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available.
We report, however, that chloroquine has strong antiviral effects on SARS-CoV infection of primate cells. These inhibitory effects are observed when the cells are treated with the drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of infection and spread of SARS CoV at clinically admissible concentrations.
Chloroquine is effective in preventing the spread of SARS CoV in cell culture. Favorable inhibition of virus spread was observed when the cells were either treated with chloroquine prior to or after SARS CoV infection. In addition, the indirect immunofluorescence assay described herein represents a simple and rapid method for screening SARS-CoV antiviral compounds.
Keywordssevere acute respiratory syndrome coronavirus chloroquine inhibition therapy
Severe acute respiratory syndrome (SARS) is an emerging disease that was first reported in Guangdong Province, China, in late 2002. The disease rapidly spread to at least 30 countries within months of its first appearance, and concerted worldwide efforts led to the identification of the etiological agent as SARS coronavirus (SARS-CoV), a novel member of the family Coronaviridae . Complete genome sequencing of SARS-CoV [2, 3] confirmed that this pathogen is not closely related to any of the previously established coronavirus groups. Budding of the SARS-CoV occurs in the Golgi apparatus  and results in the incorporation of the envelope spike glycoprotein into the virion. The spike glycoprotein is a type I membrane protein that facilitates viral attachment to the cellular receptor and initiation of infection, and angiotensin-converting enzyme-2 (ACE2) has been identified as a functional cellular receptor of SARS-CoV . We have recently shown that the processing of the spike protein was effected by furin-like convertases and that inhibition of this cleavage by a specific inhibitor abrogated cytopathicity and significantly reduced the virus titer of SARS-CoV .
Due to the severity of SARS-CoV infection, the potential for rapid spread of the disease, and the absence of proven effective and safe in vivo inhibitors of the virus, it is important to identify drugs that can effectively be used to treat or prevent potential SARS-CoV infections. Many novel therapeutic approaches have been evaluated in laboratory studies of SARS-CoV: notable among these approaches are those using siRNA , passive antibody transfer , DNA vaccination , vaccinia or parainfluenza virus expressing the spike protein [10, 11], interferons [12, 13], and monoclonal antibody to the S1-subunit of the spike glycoprotein that blocks receptor binding . In this report, we describe the identification of chloroquine as an effective pre- and post-infection antiviral agent for SARS-CoV. Chloroquine, a 9-aminoquinoline that was identified in 1934, is a weak base that increases the pH of acidic vesicles. When added extracellularly, the non-protonated portion of chloroquine enters the cell, where it becomes protonated and concentrated in acidic, low-pH organelles, such as endosomes, Golgi vesicles, and lysosomes. Chloroquine can affect virus infection in many ways, and the antiviral effect depends in part on the extent to which the virus utilizes endosomes for entry. Chloroquine has been widely used to treat human diseases, such as malaria, amoebiosis, HIV, and autoimmune diseases, without significant detrimental side effects . Together with data presented here, showing virus inhibition in cell culture by chloroquine doses compatible with patient treatment, these features suggest that further evaluation of chloroquine in animal models of SARS-CoV infection would be warranted as we progress toward finding effective antivirals for prevention or treatment of the disease.
Preinfection chloroquine treatment renders Vero E6 cells refractory to SARS-CoV infection
Postinfection chloroquine treatment is effective in preventing the spread of SARS-CoV infection
Ammonium chloride inhibits SARS-CoV infection of Vero E6 cells
Effect of chloroquine and NH4Cl on cell surface expression of ACE2
To confirm that ACE2 undergoes terminal sugar modifications and that the terminal glycosylation is affected by NH4Cl or chloroquine treatment, we performed immunopreipitation of 35S-labeled ACE2 and subjected the immunoprecipitates to neuraminidase digestion. Proteins were resolved using SDS-PAGE (Fig 5D). It is evident from the slightly faster mobility of the Golgi form of ACE2 after neuraminidase treatment (Fig 5D, compare lanes 1 and 2), that ACE2 undergoes terminal glycosylation; however, the ER form of ACE2 was not affected by neuraminidase. Cells treated with 10 μM chloroquine did not result in a significant shift; whereas 25 μM chloroquine caused the Golgi form of ACE2 to resolve similar to the neuraminidase-treated ACE2 (Fig 5D, compare lanes 5 and 6). These data provide evidence that ACE2 undergoes terminal glycosylation and that chloroquine at anti-SARS-CoV concentrations abrogates the process.
Effect of chloroquine and NH4Cl on the biosynthesis and processing of SARS-CoV spike protein
The data obtained from a 30-min pulse followed by a 3-h chase (Fig. 6B, lanes 2 and 8) confirmed our earlier observation that the SARS-CoV spike protein precursor (proS-ER) acquires Golgi-specific modifications (proS-Golgi) resulting in a ~210-kDa protein . Chloroquine at 10, 25, and 50 μM had no substantial negative impact on the appearance of the Golgi form (Fig. 6B, compare lane 2 to lanes 4–6). Only at 100 μM chloroquine was a reduction in the level of the Golgi-modified pro-spike observed (lane 3). On the other hand, NH4Cl abrogated the appearance of Golgi-modified forms at ≥10 mM (compare lane 8 with 9–11) and had a milder effect at 1 mM (lane 12). These data clearly demonstrate that the biosynthesis and proteolytic processing of SARS-CoV spike protein are not affected at chloroquine (25 and 50 μM) and NH4Cl (1 mM) doses that cause virus inhibitory effects. In addition, with 40, 20, and 10 mM NH4Cl, there was an increased accumulation of proS-ER with a concomitant decrease in the amount of oligomers (Fig. 6B, lanes 9–11). When we examined the homotrimers, we found that chloroquine at 100 μM and NH4Cl at 40 and 20 mM resulted in slightly faster mobility of the trimers (Fig. 6B, lanes 3, 9, and 10), but lower drug doses, which did exhibit significant antiviral effects, did not result in appreciable differences. These data suggest that the newly synthesized intracellular spike protein may not be a major target for chloroquine and NH4Cl antiviral action. The faster mobility of the trimer at certain higher concentration of the drugs might be due the effect of these drugs on the terminal glycosylation of the trimers.
We have identified chloroquine as an effective antiviral agent for SARS-CoV in cell culture conditions, as evidenced by its inhibitory effect when the drug was added prior to infection or after the initiation and establishment of infection. The fact that chloroquine exerts an antiviral effect during pre- and post-infection conditions suggest that it is likely to have both prophylactic and therapeutic advantages. Recently, Keyaerts et al.  reported the antiviral properties of chloroquine and identified that the drug affects SARS-CoV replication in cell culture, as evidenced by quantitative RT-PCR. Taken together with the findings of Keyaerts et al. , our analysis provides further evidence that chloroquine is effective against SARS-CoV Frankfurt and Urbani strains. We have provided evidence that chloroquine is effective in preventing SARS-CoV infection in cell culture if the drug is added to the cells 24 h prior to infection. In addition, chloroquine was significantly effective even when the drug was added 3–5 h after infection, suggesting an antiviral effect even after the establishment of infection. Since similar results were obtained by NH4Cl treatment of Vero E6 cells, the underlying mechanism(s) of action of these drugs might be similar.
Apart from the probable role of chloroquine on SARS-CoV replication, the mechanisms of action of chloroquine on SARS-CoV are not fully understood. Previous studies have suggested the elevation of pH as a mechanism by which chloroquine reduces the transduction of SARS-CoV pseudotype viruses [17, 18]. We examined the effect of chloroquine and NH4Cl on the SARS-CoV spike proteins and on its receptor, ACE2. Immunoprecipitation results of ACE2 clearly demonstrated that effective anti-SARS-CoV concentrations of chloroquine and NH4Cl also impaired the terminal glycosylation of ACE2. However, the flow cytometry data demonstrated that there are no significant differences in the cell surface expression of ACE2 in cells treated with chloroquine or NH4Cl. On the basis of these results, it is reasonable to suggest that the pre-treatment with NH4Cl or chloroquine has possibly resulted in the surface expression of the under-glycosylated ACE2. In the case of chloroquine treatment prior to infection, the impairment of terminal glycosylation of ACE2 may result in reduced binding affinities between ACE2 and SARS-CoV spike protein and negatively influence the initiation of SARS-CoV infection. Since the biosynthesis, processing, Golgi modification, and oligomerization of the newly synthesized spike protein were not appreciably affected by anti-SARS-CoV concentrations of either chloroquine or NH4Cl, we conclude that these events occur in the cell independent of the presence of the drugs. The potential contribution of these drugs in the elevation of endosomal pH and its impact on subsequent virus entry or exit could not be ruled out. A decrease in SARS-CoV pseudotype transduction in the presence of NH4Cl was observed and was attributed to the effect on intracellular pH [17, 18]. When chloroquine or NH4Cl are added after infection, these agents can rapidly raise the pH and subvert on-going fusion events between virus and endosomes, thus inhibiting the infection.
In addition, the mechanism of action of NH4Cl and chloroquine might depend on when they were added to the cells. When added after the initiation of infection, these drugs might affect the endosome-mediated fusion, subsequent virus replication, or assembly and release. Previous studies of chloroquine have demonstrated that it has multiple effects on mammalian cells in addition to the elevation of endosomal pH, including the prevention of terminal glycosyaltion of immunoglobulins . When added to virus-infected cells, chloroquine inhibited later stages in vesicular stomatitis virus maturation by inhibiting the glycoprotein expression at the cell surface , and it inhibited the production of infectious HIV-1 particles by interfering with terminal glycosylation of the glycoprotein [24, 25]. On the basis of these properties, we suggest that the cell surface expression of under-glycosylated ACE2 and its poor affinity to SARS-CoV spike protein may be the primary mechanism by which infection is prevented by drug pretreatment of cells prior to infection. On the other hand, rapid elevation of endosomal pH and abrogation of virus-endosome fusion may be the primary mechanism by which virus infection is prevented under post-treatment conditions. More detailed SARS CoV spike-ACE2 binding assays in the presence or absence of chloroquine will be performed to confirm our findings. Our studies indicate that the impact of NH4Cl and chloroquine on the ACE2 and spike protein profiles are significantly different. NH4Cl exhibits a more pronounced effect than does chloroquine on terminal glycosylation, highlighting the novel intricate differences between chloroquine and ammonium chloride in affecting the protein transport or glycosylation of SARS-CoV spike protein and its receptor, ACE2, despite their well-established similar effects of endosomal pH elevation.
The infectivity of coronaviruses other than SARS-CoV are also affected by chloroquine, as exemplified by the human CoV-229E . The inhibitory effects observed on SARS-CoV infectivity and cell spread occurred in the presence of 1–10 μM chloroquine, which are plasma concentrations achievable during the prophylaxis and treatment of malaria (varying from 1.6–12.5 μM)  and hence are well tolerated by patients. It recently was speculated that chloroquine might be effective against SARS and the authors suggested that this compound might block the production of TNFα, IL6, or IFNγ . Our data provide evidence for the possibility of using the well-established drug chloroquine in the clinical management of SARS.
Chloroquine, a relatively safe, effective and cheap drug used for treating many human diseases including malaria, amoebiosis and human immunodeficiency virus is effective in inhibiting the infection and spread of SARS CoV in cell culture. The fact that the drug has significant inhibitory antiviral effect when the susceptible cells were treated either prior to or after infection suggests a possible prophylactic and therapeutic use.
SARS-CoV infection, immunofluorescence, and immunoprecipitation analyses
Vero E6 cells (an African green monkey kidney cell line) were infected with SARS-CoV (Urbani strain) at a multiplicity of infection of 0.5 for 1 h. The cells were washed with PBS and then incubated in OPTI-MEM (Invitrogen) medium with or without various concentrations of either chloroquine or NH4Cl (both from Sigma). Immunofluorescence staining was performed with SARS-CoV-specific hyperimmune mouse ascitic fluid (HMAF)  followed by anti-mouse fluorescein-coupled antibody.
Eighteen hours after infection, the virus-containing supernatants were removed, and the cells were pulsed with 35S-(Cys) for 30 min and chased for 3 h before lysis in RIPA buffer. Clarified cell lysates and media were incubated with HMAF, and immunoprecipitated proteins were separated by 3–8% NuPAGE gel (Invitrogen); proteins were visualized by autoradiography. In some experiments, cells were chased for 3 h with isotope-free medium. Clarified cell supernatants were also immunoprecipitated with SARS-CoV-specific HMAF.
ACE2 flow cytometry analysis and biosynthesis
Vero E6 cells were seeded in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum. The next day, the cells were incubated in Opti-MEM (Invitrogen) in the presence or absence of 10 μM chloroquine or 20 mM NH4Cl. To analyze the levels of ACE2 at the cell surface, cells were incubated on ice with 10 μg/mL affinity-purified goat anti-ACE2 antibody (R&D Systems) and then incubated with FITC-labeled swine anti-goat IgG antibody (Caltag Laboratories). Labeled cells were analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences). For ACE2 biosynthesis studies, Vero E6 cells were pulsed with 250 μCi 35S-(Met) (Perkin Elmer) for 3 h with the indicated concentrations of chloroquine or NH4Cl and then lysed in RIPA buffer. Clarified lysates were immunoprecipitated with an affinity-purified goat anti-ACE2 antibody (R&D systems), and the immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis.
We thank Claudia Chesley and Jonathan Towner for critical reading of the manuscript. This work was supported by a Canadian PENCE grant (T3), CIHR group grant #MGC 64518, and CIHR grant #MGP-44363 (to NGS).
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