Chloroquine is a potent inhibitor of SARS coronavirus

CDC in 2005: Chloroquine is a potent inhibitor of SARS coronavirus infection and spread

CDC

Martin J Vincent, Eric Bergeron, Suzanne Benjannet, Bobbie R Erickson, Pierre E Rollin, Thomas G Ksiazek, Nabil G Seidah and Stuart T Nichol*

Address: Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, Georgia, 30333, USA and Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, QCH2W1R7, Canada

Email: Martin J Vincent – mvincent@cdc.gov; Eric Bergeron – bergere@ircm.qc.ca; Suzanne Benjannet – benjans@ircm.qc.ca; Bobbie R Erickson – BErickson1@cdc.gov; Pierre E Rollin – PRollin@cdc.gov; Thomas G Ksiazek – TKsiazek@cdc.gov; Nabil G Seidah – seidahn@ircm.qc.ca;

Stuart T NicholSNichol@cdc.gov is the corresponding author of severe acute respiratory syndrome coronavirus chloroquine inhibition therapy

Dr. Gorter states that his conclusion is that this study (already published in August of 2005), and several clinical studies since then, prove the efficacy of Chloroquine in the prophylaxis and treatment of SARS-CoV-2 infection.

That even the Lancet tried to nullify these publications in summer 2020 but was then forced to retract the articles show that one can only come to one conclusion:

To obtain a temporary, emergency approval to conduct clinical studies in humans with a novel substance that has hardly been studied before, one must meet two main criteria:

  • A life-threatening disease or a pandemic with huge numbers of deaths
  • No known medications available

Is it, therefore, that the CDC in the USA and the WHO declared that no therapy exists for SARS-CoV-2 (COVID-19) and thus, the Pharmaceutical Industry could rapidly obtain approval to expose hundreds of millions of people around the world to an experimental (mRNA) vaccine?

In the 2000s and in large animals, mRNA vaccines were tried but usually, these trials were aborted due to significant (even fatal) side effects.

As a clinical researcher, Dr. Gorter argues that many of these autoimmune-like diseases after mRNA injections were very similar to prison-based diseases (like “Mad Cow Disease, Scrapie and neurological diseases in humans like Parkinson and MS.”)

 More and more data are surfacing that prions are caused by “glued together” double RNS molecules. To this subject, Dr. Gorter published a review article with more than fifty referrals to world-leading researchers who –in his opinion- prove that the source of prions is folded RNA molecules.

http://robert-gorter.info/rnas-behave-like-prions/

Chloroquine is a very safe, effective, and cheap drug that is being used for treating many human diseases including malaria, amoebiasis, several autoimmune diseases, and human immunodeficiency virus (HIV) and is effective in inhibiting the infection and spread of SARS CoV in cell culture and in humans. The fact that the drug has a significant inhibitory antiviral effect when the susceptible cells were treated either prior to or after infection strongly suggests its prophylactic and therapeutic use.

At the bottom of this article, one can find the link to the complete article published by the CDC in August 2005 and other information.

https://stacks.cdc.gov/view/cdc/3620

Abstract

Background: Severe acute respiratory syndrome  (SARS)  is  caused by  a  newly discovered coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available.

Results: 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.

Conclusion:

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.

Background:

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 (1). Complete genome  sequencing  of  SARS-CoV  (2,3)  confirmed  that this   pathogen   is   not   closely   related   to   any   of   the

Published: 22 August 2005 Virology Journal 2005, 2:69doi:10.1186/1743-422X-2-69 Received: 12 July 2005 Accepted: 22 August 2005. This article is available from: http://www.virologyj.com/content/2/1/69 2005 Vincent et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Virology Journal 2005:  http://www.virologyj.com/content/2/1/69 Page 2 of 10 (page number not for citation purposes) previously established coronavirus groups. Budding of the SARS-CoV occurs in the Golgi apparatus (4) 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-con-verting  enzyme-2  (ACE2)  has  been  identified  as  a  functional cellular receptor of SARS-CoV (5). 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 (6).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 impor-tant to identify drugs that can effectively be used to treator  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 (7), passive antibody transfer (8), DNA vaccination (9), vaccinia or parainfluenza virus expressing the spike protein (10,11), interferons (12,13), and mono-clonal antibody to the S1-subunit of the spike glycoprotein that blocks receptor binding (14). 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  become sprotonated    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, amoebiasis,  HIV,  and  autoimmune  diseases, without significant detrimental side effects (15). 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.

Results

Pre-infection 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  pre-treated 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-dependent decrease in virus antigen-positive cells was observed starting at 0.1 μM chloroquine, and concentrations of 10 μM completely abolished SARS-CoVinfection.  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. Post infection 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, VeroE6  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-dependent  decrease  in  the  number  of virus antigen-positive cells (Fig. 2A). As little as 0.1–1 μ Mchloroquine 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. 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  fur-ther extended the analysis by adding chloroquine 3 and 5h 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).

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  lysosome-tropic  agent,  NH4Cl,  might also  function  in  a  similar  manner.  Ammonium  chloride has  been  widely  used  in  studies  addressing  endosome-mediated  virus  entry.  Coincidently,  NH4Cl  was  recently shown  to  reduce  the  transduction  of  pseudo type  viruses decorated  with  SARS-CoV  spike  protein  [17,18].  In anattempt to examine if NH4Cl 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   NH4Cl.   In   both   cases,   we observed a 93–99%  inhibition  with  NH4Cl  at  ≥  5  mM. These data indicated  that  NH4Cl  (≥  5  mM)  and  chloroquine (≥ 10 μM) are very effective in reducing SARS-CoV infection. These results suggest that effects of chloroquine and NH4Cl in controlling SARS CoV infection and spread might be mediated by similar mechanism(s).Effect of chloroquine and NH4Cl on cell surface expression of ACE2We  performed  additional  experiments  to  elucidate  the mechanism of SARS-CoV inhibition by chloroquine andNH4Cl.  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.  Flowcytometry  analysis  was  performed  on  Vero  E6  cells  that were either untreated or treated with highly effective anti-SARS-CoV concentrations of chloroquine or NH4Cl. 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  Prophylactic effects of chloroquine 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.

We also examined the terminal glycosylation status of ACE2 when the cells were treated with chloroquine (Fig.  5C).  Similar  to  NH4Cl,  a  stepwise  increase  in  the electrophoretic  mobility  of  ACE2  was  observed  within creasing 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. Timed post-infection treatment with chloroquine (Figure 3). 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.NH4Cl inhibits SARS-CoV during pre or post infection treatment Figure 4NH4Cl inhibits SARS-CoV during pre or post infection treatment. NH4Cl 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.

Tt  can  be  inferred  that  NH4Cl  and  chloroquine  both impaired   the   terminal   glycosylation   of   ACE2,   whileNH4Cl resulted in a more dramatic effect. Although ACE2is  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  NH4Cl  and chloroquine. To confirm that ACE2 undergoes terminal sugar modifications and  that  the  terminal  glycosylation  is  affected  byNH4Cl or chloroquine treatment, we performed immuno-precipitation of 35S-labeled ACE2 and subjected the immune effects of lysomotropic agents on the cell-surface expression and biosynthesis of ACE2Figure 5Effect 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 NH4Cl (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 NH4Cl. Vero E6 cells were pulse-labeled for 3 h with 35S-Met, and the cell lysates were immunoprecipitated with an ACE2 antibody (lane 1). Pre-incubation 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 NH4Cl 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.

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 asignificant  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. Effects of chloroquine and NH4Cl on the biosynthesis and processing of SARS-CoV spike protein. We  next  addressed  whether  the  lysosomotropic  drugs (NH4Cl  and  chloroquine)  affect  the  biosynthesis,  glycosylation, and/or trafficking of the SARS-CoV spike glycoprotein. For this purpose, Vero E6 cells were infected withSARS-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-minpulse 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), therewas   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 effects of NH4Cl and chloroquine (CQ) on the biosynthesis, processing, and glycosylation of SARS-CoV spike protein (Figure 6). Effects of NH4Cl 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 NH4Cl 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 immuneprecipitated with HMAF. Virus proteins were resolved using 3–8% Nu PAGE gel (Invitro-gen). 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. Pro-S-ER and pro-S-Golgi are the pro-spike of SARS-Co in the ER and Golgi compartments, respectively and pro-S-ungly is the ungly cosylated pro-spike ER.

The biosynthesis  and  processing  of  the  spike  glycoprotein  in the  presence  of  chloroquine  concentrations  (10  and  50μM) 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 withNH4Cl, and the data suggested that the biosynthesis and processing  of  the  spike  protein  were  also  not  negatively affected by NH4Cl (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  NH4Cl  (Fig.  6A,  lanes  3,  9,  and  10) were  slightly  lower  than  in  control  cells  or  cells  treated with lower drug concentrations. The data obtained from a 30-min pulse followed by a 3-hchase (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  [6].  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 lane8 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  (1mM) 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 at100 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  andNH4Cl 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 thetrimers.

Discussion

We  have  identified  chloroquine  as  an  effective  anti-viral 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. (21) reported the anti-viral  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. (21]) 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 3–5h 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 under-lying  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 pseudo type  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  flowcytometry 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  orNH4Cl,  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  pseudo-typetransduction 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 effusion 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  (22).  When added to virus-infected cells, chloroquine inhibited later stages in vesicular stomatitis virus maturation by inhibiting  the  glycoprotein  expression  at  the  cell  surface  (23),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-glyco-sylated 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   CoVspike-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,  high-lighting  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 (15). 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) [26] 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 (15). Our data provide evidence for the  possibility  of  using  the  well-established drug chloroquine in the clinical management of SARS.

Conclusion

Chloroquine is a very safe,  effective  and  cheap  drug used for treating many human diseases including malaria, amoebiasis, several autoimmune diseases and human immunodeficiency virus (HIV) 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  strongly suggests  a  possible  prophylactic and therapeutic use.

Methods

SARS-CoV infection, immunofluorescence, and immunoprecipitation analysesVero 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)  (8)  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 immune-precipitated 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  immune-precipitated  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 10μM 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 FAC-S Calibur flow cytometer (BD Biosciences). For ACE2 bio-synthesis studies, Vero E6 cells were pulsed with 250 μCi35S-(Met) (Perkin Elmer) for 3 h with the indicated concentrations  of  chloroquine  or  NH4Cl  and  then  lysed  in RIPA  buffer.  Clarified lysates were immune-precipitated with an affinity-purified  goat  anti-ACE2  antibody  (R&Dsystems), and the immune-precipitated proteins were separated by SDS-polyacrylamide gel electrophoresis.

 

https://stacks.cdc.gov/view/cdc/3620

 

 

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