Preinfection chloroquine treatment renders Vero E6 cells refractory to SARS-CoV infection

In order to investigate if chloroquine might prevent SARS-CoV infection, permissive Vero E6 cells [1] were pretreated with various concentrations of chloroquine (0.1–10 μM) for 20–24 h prior to virus infection. Cells were then infected with SARS-CoV, and virus antigens were visualized by indirect immunofluorescence as described in Materials and Methods. Microscopic examination (Fig. 1A) of the control cells (untreated, infected) revealed extensive SARS-CoV-specific immunostaining of the monolayer. A dose-dependant decrease in virus antigen-positive cells was observed starting at 0.1 μM chloroquine, and concentrations of 10 μM completely abolished SARS-CoV infection. For quantitative purposes, we counted the number of cells stained positive from three random locations on a slide. The average number of positively stained control cells was scored as 100% and was compared with the number of positive cells observed under various chloroquine concentrations (Fig. 1B). Pretreatment with 0.1, 1, and 10 μM chloroquine reduced infectivity by 28%, 53%, and 100%, respectively. Reproducible results were obtained from three independent experiments. These data demonstrated that pretreatment of Vero E6 cells with chloroquine rendered these cells refractory to SARS-CoV infection.

Figure 1 Prophylactic effect of chloroquine. Vero E6 cells pre-treated with chloroquine for 20 hrs. Chloroquine-containing media were removed and the cells were washed with phosphate buffered saline before they were infected with SARS-CoV (0.5 multiplicity of infection) for 1 h. in the absence of chloroquine. Virus was then removed and the cells were maintained in Opti-MEM (Invitrogen) for 16–18 h in the absence of chloroquine. SARS-CoV antigens were stained with virus-specific HMAF, followed by FITC-conjugated secondary antibodies. (A) The concentration of chloroquine used is indicated on the top of each panel. (B) SARS-CoV antigen-positive cells at three random locations were captured by using a digital camera, the number of antigen-positive cells was determined, and the average inhibition was calculated. Percent inhibition was obtained by considering the untreated control as 0% inhibition. The vertical bars represent the range of SEM. Full size image

Postinfection chloroquine treatment is effective in preventing the spread of SARS-CoV infection

In order to investigate the antiviral properties of chloroquine on SARS-CoV after the initiation of infection, Vero E6 cells were infected with the virus and fresh medium supplemented with various concentrations of chloroquine was added immediately after virus adsorption. Infected cells were incubated for an additional 16–18 h, after which the presence of virus antigens was analyzed by indirect immunofluorescence analysis. When chloroquine was added after the initiation of infection, there was a dramatic dose-dependant decrease in the number of virus antigen-positive cells (Fig. 2A). As little as 0.1–1 μM chloroquine reduced the infection by 50% and up to 90–94% inhibition was observed with 33–100 μM concentrations (Fig. 2B). At concentrations of chloroquine in excess of 1 μM, only a small number of individual cells were initially infected, and the spread of the infection to adjacent cells was all but eliminated. A half-maximal inhibitory effect was estimated to occur at 4.4 ± 1.0 μM chloroquine (Fig. 2C). These data clearly show that addition of chloroquine can effectively reduce the establishment of infection and spread of SARS-CoV if the drug is added immediately following virus adsorption.

Figure 2 Post-infection chloroquine treatment reduces SARS-CoV infection and spread. Vero E6 cells were seeded and infected as described for Fig. 1 except that chloroquine was added only after virus adsorption. Cells were maintained in Opti-MEM (Invitrogen) containing chloroquine for 16–18 h, after which they were processed for immunofluorescence. (A) The concentration of chloroquine is indicated on the top. (B) Percent inhibition and SEM were calculated as in Fig. 1B. (C) The effective dose (ED 50 ) was calculated using commercially available software (Grafit, version 4, Erithacus Software). Full size image

Electron microscopic analysis indicated the appearance of significant amounts of extracellular virus particles 5–6 h after infection [16]. Since we observed antiviral effects by chloroquine immediately after virus adsorption, we further extended the analysis by adding chloroquine 3 and 5 h after virus adsorption and examined for the presence of virus antigens after 20 h. We found that chloroquine was still significantly effective even when added 5 h after infection (Fig. 3); however, to obtain equivalent antiviral effect, a higher concentration of chloroquine was required if the drug was added 3 or 5 h after adsorption.

Figure 3 Timed post-infection treatment with chloroquine. This experiment is similar to that depicted in Fig. 2 except that cells were infected at 1 multiplicity of infection, and chloroquine (10, 25, and 50 μM) was added 3 or 5 h after infection. Full size image

Ammonium chloride inhibits SARS-CoV infection of Vero E6 cells

Since chloroquine inhibited SARS-CoV infection when added before or after infection, we hypothesized that another common lysosomotropic agent, NH 4 Cl, might also function in a similar manner. Ammonium chloride has been widely used in studies addressing endosome-mediated virus entry. Coincidently, NH 4 Cl was recently shown to reduce the transduction of pseudotype viruses decorated with SARS-CoV spike protein [17, 18]. In an attempt to examine if NH 4 Cl functions similarly to chloroquine, we performed infection analyses in Vero E6 cells before (Fig. 4A) and after (Fig. 4B) they were treated with various concentrations of NH 4 Cl. In both cases, we observed a 93–99% inhibition with NH 4 Cl at ≥ 5 mM. These data indicated that NH 4 Cl (≥ 5 mM) and chloroquine (≥ 10 μM) are very effective in reducing SARS-CoV infection. These results suggest that effects of chloroquine and NH 4 Cl in controlling SARS CoV infection and spread might be mediated by similar mechanism(s).

Figure 4 NH 4 Cl inhibits SARS-CoV during pre or post infection treatment. NH 4 Cl was added to the cells either before (A) or after (B) infection, similar to what was done for chloroquine in Figs 1 and 2. Antigen-positive cells were counted, and the results were presented as in Fig. 1B. Full size image

Effect of chloroquine and NH 4 Cl on cell surface expression of ACE2

We performed additional experiments to elucidate the mechanism of SARS-CoV inhibition by chloroquine and NH 4 Cl. Since intra-vesicular acidic pH regulates cellular functions, including N-glycosylation trimming, cellular trafficking, and various enzymatic activities, it was of interest to characterize the effect of both drugs on the processing, glycosylation, and cellular sorting of SARS-CoV spike glycoprotein and its receptor, ACE2. Flow cytometry analysis was performed on Vero E6 cells that were either untreated or treated with highly effective anti-SARS-CoV concentrations of chloroquine or NH 4 Cl. The results revealed that neither drug caused a significant change in the levels of cell-surface ACE2, indicating that the observed inhibitory effects on SARS-CoV infection are not due to the lack of available cell-surface ACE2 (Fig. 5A). We next analyzed the molecular forms of endogenous ACE2 in untreated Vero E6 cells and in cells that were pre-incubated for 1 h with various concentrations of either NH 4 Cl (2.5–10 mM) or chloroquine (1 and 10 μM) and labeled with 35S-(Met) for 3 h in the presence or absence of the drugs (Fig. 5B and 5C). Under normal conditions, we observed two immunoreactive ACE2 forms, migrating at ~105 and ~113 kDa, respectively (Fig. 5B, lane 1). The ~105-kDa protein is endoglycosidase H sensitive, suggesting that it represents the endoplasmic reticulum (ER) localized form, whereas the ~113-kDa protein is endoglycosidase H resistant and represents the Golgi-modified form of ACE2 [19]. The specificity of the antibody was confirmed by displacing the immunoreactive protein bands with excess cold-soluble human recombinant ACE2 (+ rhACE2; Fig. 5B, lane 2). When we analyzed ACE2 forms in the presence of NH 4 Cl, a clear stepwise increase in the migration of the ~113-kDa protein was observed with increasing concentrations of NH 4 Cl, with a maximal effect observed at 10 mM NH 4 Cl, resulting in only the ER form of ACE2 being visible on the gel (Fig. 5B, compare lanes 3–5). This suggested that the trimming and/or terminal modifications of the N-glycosylated chains of ACE2 were affected by NH 4 Cl treatment. In addition, at 10 mM NH 4 Cl, the ER form of ACE2 migrated with slightly faster mobility, indicating that NH 4 Cl at that concentration might also affect core glycosylation. We also examined the terminal glycosylation status of ACE2 when the cells were treated with chloroquine (Fig. 5C). Similar to NH 4 Cl, a stepwise increase in the electrophoretic mobility of ACE2 was observed with increasing concentrations of chloroquine. At 25 μM chloroquine, the faster electrophoretic mobility of the Golgi-modified form of ACE2 was clearly evident. On the basis of the flow cytometry and immunoprecipitation analyses, it can be inferred that NH 4 Cl and chloroquine both impaired the terminal glycosylation of ACE2, while NH 4 Cl resulted in a more dramatic effect. Although ACE2 is expressed in similar quantities at the cell surface, the variations in its glycosylation status might render the ACE2-SARS-CoV interaction less efficient and inhibit virus entry when the cells are treated with NH 4 Cl and chloroquine.

Figure 5 Effect of lysomotropic agents on the cell-surface expression and biosynthesis of ACE2. (A) Vero E6 cells were cultured for 20 h in the absence (control) or presence of chloroquine (10 μM) or NH 4 Cl (20 mM). Cells were labeled with anti-ACE2 (grey histogram) or with a secondary antibody alone (white histogram). (B) Biosynthesis of ACE2 in untreated cells or in cells treated with NH 4 Cl. Vero E6 cells were pulse-labeled for 3 h with 35S-Met, and the cell lysates were immunoprecipitated with an ACE2 antibody (lane 1). Preincunbation of the antibody with recombinant human ACE2 (rhACE2) completely abolished the signal (lane 2). The positions of the endoglycosidase H-sensitive ER form and the endoglycosidase H-resistant Golgi form of ACE2 are emphasized. Note that the increasing concentration of NH 4 Cl resulting in the decrease of the Golgi form of ACE2. (C) A similar experiment was performed in the presence of the indicated concentrations of chloroquine. Note the loss of terminal glycans with increasing concentrations of chloroquine. (D) The terminal glycosidic modification of ACE2 was evaluated by neuraminidase treatment of immunoprecipitated ACE2. Here cells were treated with 1–25 μM concentrations of chloroquine during starvation, pulse, and 3-h chase. Full size image

To confirm that ACE2 undergoes terminal sugar modifications and that the terminal glycosylation is affected by NH 4 Cl 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 NH 4 Cl on the biosynthesis and processing of SARS-CoV spike protein

We next addressed whether the lysosomotropic drugs (NH 4 Cl and chloroquine) affect the biosynthesis, glycosylation, and/or trafficking of the SARS-CoV spike glycoprotein. For this purpose, Vero E6 cells were infected with SARS-CoV for 18 h. Chloroquine or ammonium chloride was added to these cells during while they were being starved (1 h), labeled (30 min) or chased (3 h). The cell lysates were analyzed by immunoprecipitation with the SARS-specific polyclonal antibody (HMAF). The 30-min pulse results indicated that pro-spike (proS) was synthesized as a ~190-kDa precursor (proS-ER) and processed into ~125-, ~105-, and ~80-kDa proteins (Fig. 6A, lane 2), a result identical to that in our previous analysis [6]. Except for the 100 μM chloroquine (Fig. 6A, lane 3), there was no significant difference in the biosynthesis or processing of the virus spike protein in untreated or chloroquine-treated cells (Fig. 6A, lanes 4–6). It should be noted that chloroquine at 100 μM resulted in an overall decrease in biosynthesis and in the levels of processed virus glycoprotein. In view of the lack of reduction in the biosynthesis and processing of the spike glycoprotein in the presence of chloroquine concentrations (10 and 50 μM) that caused large reductions in SARS-CoV replication and spread, we conclude that the antiviral effect is probably not due to alteration of virus glycoprotein biosynthesis and processing. Similar analyses were performed with NH 4 Cl, and the data suggested that the biosynthesis and processing of the spike protein were also not negatively affected by NH 4 Cl (Fig. 6A, lanes 7–12). Consistent with our previous analysis [6], we observed the presence of a larger protein, which is referred to here as oligomers. Recently, Song et al. [20] provided evidence that these are homotrimers of the SARS-CoV spike protein and were incorporated into the virions. Interestingly, the levels of the homotrimers in cells treated with 100 μM chloroquine and 40 and 20 mM NH 4 Cl (Fig. 6A, lanes 3, 9, and 10) were slightly lower than in control cells or cells treated with lower drug concentrations.

Figure 6 Effects of NH 4 Cl and chloroquine (CQ) on the biosynthesis, processing, and glycosylation of SARS-CoV spike protein. Vero E6 cells were infected with SARS-CoV as described in Fig. 2. CQ or NH 4 Cl was added during the periods of starvation (1 h) and labeling (30 min) with 35S-Cys and followed by chase for 3 h in the presence of unlabeled medium. Cells were lysed in RIPA buffer and immunoprecipitated with HMAF. Virus proteins were resolved using 3–8% NuPAGE gel (Invitrogen). The cells presented were labeled for 30 min (A) and chased for 3 h (B). The migration positions of the various spike molecular forms are indicated at the right side, and those of the molecular standards are shown to the left side. proS-ER and proS-Golgi are the pro-spike of SARS-Co in the ER and Golgi compartments, respectively and proS-ungly is the unglycosylated pro-spike ER. Full size image