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1 Title: Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway Authors: Chaitra Prabhakara 1,* , Rashmi Godbole 1,2,* , Parijat Sil 1,* , Sowmya Jahnavi 1,* , Thomas S van Zanten 1,# , Dhruv Sheth 1,# , Neeraja Subhash 1 , Anchal Chandra 1 , Vijay Kumar Nuthakki 4 , Theja Parassini Puthiyapurayil 3 , Riyaz Ahmed 4 , Ashaq Hussain Najar 4 , Sai Manoz Lingamallu 3,5 , Snigdhadev Das 1 , Bhagyashri Mahajan 1 , Praveen Vemula 3 , Sandip B Bharate 4 , Parvinder Pal Singh 4 , Ram Vishwakarma 4 , Arjun Guha 3 , Varadharajan Sundaramurthy 1 and Satyajit Mayor 1 Affiliations: 1- National Centre for Biological Sciences (TIFR), Bengaluru, India 2- University of Trans-Disciplinary Health Sciences and Technology (TDU), Bengaluru, India 3- Institute for Stem Cell Science and Regenerative Medicine (inSTEM), Bengaluru, India 4- CSIR - Indian Institute of Integrative Medicine, Jammu, India 5- Manipal Academy of Higher Education (MAHE), Madhav Nagar, Manipal, Karnataka, India * and # contributed equally Correspondence to: [email protected] preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529 doi: bioRxiv preprint
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Title: Niclosamide inhibits SARS-CoV2 entry by blocking ...Dec 16, 2020  · 3 44 reduce MERS, SARS-CoV, SARS-Cov2 infection 24,28,29.These studies emphasize the importance of a 45

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  • 1

    Title: Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent

    CLIC/GEEC endocytic pathway

    Authors:

    Chaitra Prabhakara1,*, Rashmi Godbole1,2,*, Parijat Sil1,*, Sowmya Jahnavi1,*, Thomas S van Zanten1,#,

    Dhruv Sheth1,#, Neeraja Subhash1, Anchal Chandra1, Vijay Kumar Nuthakki4, Theja Parassini

    Puthiyapurayil3, Riyaz Ahmed4, Ashaq Hussain Najar4, Sai Manoz Lingamallu3,5, Snigdhadev Das1,

    Bhagyashri Mahajan1, Praveen Vemula3, Sandip B Bharate4, Parvinder Pal Singh4, Ram Vishwakarma4,

    Arjun Guha3, Varadharajan Sundaramurthy1 and Satyajit Mayor1

    Affiliations:

    1- National Centre for Biological Sciences (TIFR), Bengaluru, India

    2- University of Trans-Disciplinary Health Sciences and Technology (TDU), Bengaluru, India

    3- Institute for Stem Cell Science and Regenerative Medicine (inSTEM), Bengaluru, India

    4- CSIR - Indian Institute of Integrative Medicine, Jammu, India

    5- Manipal Academy of Higher Education (MAHE), Madhav Nagar, Manipal, Karnataka, India

    * and # contributed equally

    Correspondence to: [email protected]

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    mailto:[email protected]://doi.org/10.1101/2020.12.16.422529

  • 2

    Abstract: 1

    Many viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of 2

    SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. 3

    Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the 4

    clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal 5

    acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block 6

    the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that 7

    these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD 8

    uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a 9

    pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification 10

    inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, 11

    could provide broader applicability in subverting infection of similar category viruses entering host 12

    cells via this pH-dependent endocytic pathway. 13

    Keywords: SARS-CoV2 entry, CLIC/GEEC endocytosis, Spike-pseudovirus, Endosomal acidification 14

    inhibitors, Niclosamide, Chloroquine 15

    Introduction: 16

    Coronaviruses (CoVs) are a group of related enveloped RNA viruses of which two alpha CoVs (229E 17

    and NL63) and four beta CoVs (OC43, HKU, SARS, and MERS) are known to cause respiratory tract 18

    infections in humans. The recent emergence of SARS-CoV2 and its rapid spread across the world has 19

    posed a global health emergency 1. While several therapeutic strategies are currently being used to 20

    alleviate the respiratory symptoms of patients infected with SARS-CoV2 2,3, there is limited 21

    understanding of the cell biology of viral entry as well as the availability of drugs which target this 22

    process. A search for antivirals affecting the endocytic entry of viruses is particularly exciting as 23

    infections from multiple related viruses can be controlled through the inhibition of a common step. 24

    Virus entry into host cells is a multistep process. A key step in successful invasion is the release of viral 25

    genomic content into the host cell cytoplasm. To achieve this, viruses bind to specific cell surface 26

    receptors and subsequently undergo membrane fusion either directly at the plasma membrane or 27

    following endocytic uptake. While fusion directly at the plasma membrane is well established for HIV 28

    and Influenza virus infections 4,5, both alternatives of entry are feasible for CoV infections depending 29

    on the availability of receptors and proteases at the host cell surface. Different CoVs interact with a 30

    range of specific receptors for entry. For instance, CoV 229E binds to CD13 (aminopeptidase N) 6, 31

    CoVs OC43 and HKU1 recognize 9-O-acetylated sialic acids 7, MERS uses DPP4/CD26 8 and CoVs 32

    NL63 9, SARS-CoV 10 and SARS-CoV2 11 interact with angiotensin converting enzyme 2 (ACE2). 33

    Although ACE2 is a well studied receptor, other receptors for SARS-CoV2 are being discovered 12–16. 34

    Additionally, CoVs require proteolytic processing of the viral envelope spike protein by host cell 35

    proteases to gain entry 17,18. Therefore, these viruses can directly fuse at the cell surface if the Spike 36

    protein is cleaved by a cell surface serine protease like TMPRSS2 11,19, or utilize an endo-lysosomal 37

    route for fusion, where the Spike protein is primed by cysteine protease Cathepsins 11,20–22. 38

    The role of the endo-lysosomal network appears to be crucial in delivering these viruses to acidic 39

    compartments. Cathepsins function optimally in a low pH environment 17,23. Inhibitors of acidification 40

    which increase the pH of endosomal compartments significantly reduce the infection of spike 41

    pseudotyped as well as native MERS-CoV, SARS-CoV, SARS-CoV2 viruses 11,24–27. Supporting this 42

    view, drugs inhibiting the maturation of late endosome to lysosome, Apilimod and YM201636, also 43

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.16.422529

  • 3

    reduce MERS, SARS-CoV, SARS-Cov2 infection 24,28,29. These studies emphasize the importance of a 44

    pH-dependent endocytic route in viral entry and infection. However, the endocytic routes preferred by 45

    SARS-CoV2 for host cell entry are largely unknown. 46

    Multiple endocytic pathways operate at the cell surface 30,31. One of these, the clathrin and dynamin 47

    independent CLIC/GEEC (CG) endocytic pathway 32, is of particular interest here as uptake through 48

    this pathway is known to be pH-dependent. Vacuolar ATPases (V-ATPases), which actively pump 49

    protons into the endocytic compartments 33, play a crucial role in the formation of CG endosomes as 50

    established using genetic and pharmacological perturbations 34,35. By contrast, uptake through clathrin 51

    mediated endocytosis (CME) remains unaltered upon V-ATPase perturbation 34. The homotypic fusion 52

    of nascent CG endosomes (called CLICs – clathrin-independent carriers) forms highly acidic early 53

    endosomal compartments of the CG pathway (called GEECs – GPI anchored protein enriched early 54

    endosomal compartments) with an estimated luminal pH of 6.0 36. Thus, GEECs could provide a 55

    conducive environment for viral uncoating and membrane fusion. Additionally, V-ATPases and 56

    ARP2/3 complex, both imperative for CG endocytosis 37, are identified as host factors necessary for 57

    SARS-CoV2 viral infection in genome-wide loss of function screen 38. Interestingly, Adeno-associated 58

    virus (AAV2) hijacks the CG pathway for infection 39 and SARS-CoV has also been reported to enter 59

    cells through a clathrin and dynamin independent endocytic pathway 26. These observations prompted 60

    us to study the role of CG endocytosis in the context of SARS-CoV2 entry and infection. 61

    In this report, we show that the receptor binding domain (RBD) of SARS-CoV2 Spike protein is 62

    endocytosed through the CG pathway and its uptake is sensitive to pharmacological perturbations of 63

    the CG pathway. RBD uptake, similar to CG cargo uptake, is strongly inhibited by acidification 64

    inhibitors such as BafilomycinA1 and NH4Cl. Inhibitors of endosomal acidification also blocked 65

    infection by SARS-CoV2 Spike-pseudoviruses. Extending our observations, we conducted a targeted 66

    screen using a subset of FDA-approved drugs which are known to interfere with endosomal 67

    acidification. We identified Niclosamide as a promising candidate that inhibits RBD uptake, Spike-68

    pseudovirus infection and, in combination, potentiates the effects of Hydroxychloroquine. We suggest 69

    that Niclosamide could be a feasible start point for developing small molecule entry inhibitors to 70

    mitigate SARS-CoV2 infection. 71

    Results: 72

    Generation of SARS-CoV2 probe to study its endocytosis itinerary 73

    The Spike (S) protein of SARS-CoV2 plays crucial roles in mediating viral entry to cells. S protein 74

    binds to the receptors on the host cell surface through the S1 subunit which harbours the receptor 75

    binding domain (RBD) and aids in membrane fusion through the S2 subunit 40. To explore the 76

    trafficking route of SARS-CoV2 in human cells, we purified RBD protein, following a previously 77

    established method 41, and generated fluorescently labelled RBD using NHS-ester chemistry (Figure 78

    S1A, S1B, Methods). We chose human adenocarcinoma gastric cells (AGS cells) as a model system to 79

    study RBD uptake as the cell line exhibits multiple endocytic routes 42 and is also permissive to infection 80

    by SARS-CoV2 Spike-pseudovirus (Figure S6E). We tested the specificity of the labelled RBD probe 81

    in AGS cells transiently overexpressing myc-tagged ACE2 and found that more RBD was bound to 82

    cells overexpressing ACE2 (Figure S1C). We observed a positive correlation between the amount of 83

    RBD endocytosed and surface levels of ACE2 (Figure S1D, S1E), supporting the notion that ACE2 is 84

    one of the cell surface receptors of RBD 11. 85

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.16.422529

  • 4

    RBD is internalized via CG endocytosis and RBD uptake is sensitive to CG Pathway inhibitors 86

    We employed the methodology of tracking RBD uptake along with cargoes specific to CME 87

    (transferrin) and CG (10kDa dextran) endocytic pathway to determine the endocytic route taken up by 88

    RBD (Figure 1A). AGS cells without overexpression of ACE2 also support RBD uptake suggesting 89

    that there is sufficient endogenous receptor expressed in these cells. At 10 minutes post internalization, 90

    transferrin endosomes of the CME pathway are distinct from dextran endosomes of the CG pathway. 91

    At these times, internalized RBD is colocalized with endosomes containing the CG cargo but not the 92

    CME cargo (Figure S2A, S2B). At 30 minutes post internalization, as well, this segregation remains. 93

    While a small fraction of RBD endosomes were colocalized with endosomes containing both transferrin 94

    and dextran, a large fraction of RBD endosomes were localized to compartments uniquely marked by 95

    dextran (Figure 1B, 1C; compare % RBD with transferrin and dextran). This suggests that the itinerary 96

    of uptake of RBD is similar to CG cargo and different from CME cargo. 97

    Towards determining the trafficking route of RBD, we examined the effect of inhibitors of CG pathway 98

    on RBD, dextran and transferrin uptake in AGS cells. Cells were subjected to a brief pre-treatment with 99

    different inhibitors (30 minutes), followed by a pulse of RBD, dextran and transferrin (30 minutes) in 100

    the presence of these inhibitors (Methods). CG pathway is regulated by small GTPases - CDC42 43, 101

    Arf1 44 and GEF of Arf1, GBF1 45. Inhibitors that block the function of these regulators affect the 102

    formation of CG endosomes without altering uptake through the CME pathway. The inhibitor AN96, 103

    which is a stable analog of LG-186 46,47, targets GBF1 and specifically affects the CG pathway (Godbole 104

    et al., Manuscript in preparation). We observed that AN96 treatment reduced both RBD and dextran 105

    uptake but had minimal effects on the amount of transferrin internalized (Figure 1D, 1E). We also 106

    observed that the peri-nuclear transferrin recycling endosomal pool was redistributed throughout the 107

    cytoplasm upon treatment with AN96 without affecting the net amount of transferrin internalized. 108

    Another CG pathway inhibitor, ML141 (CDC42 inhibitor) 47, also significantly decreased both dextran 109

    as well as RBD uptake (Figure S2E, S2F). 110

    Blocking of the CG pathway often results in the redistribution of CG cargo towards CME pathway 42. 111

    Therefore, using high-resolution imaging, we assessed the fate of the RBD endosomes that continued 112

    to be internalized upon treatment with AN96. We observed that an increased fraction of internalized 113

    RBD endosomes colocalized with transferrin in AN96 treated cells compared to that of control (Figure 114

    S2C, S2D(i)). Similarly, increased co-occurrence was observed in the fraction of dextran endosomes 115

    associated with transferrin endosomes on comparing the control with AN96 treated cells (Figure 116

    S2D(iii)). Blocking the CG pathway results in altered trafficking itinerary of RBD and increases its 117

    association with transferrin. This suggests that RBD could be redirected to be internalized via the CME 118

    upon blocking the CG pathway. 119

    Since 10kDa dextran marks both CG cargo as well as larger endocytic compartments like those derived 120

    from macropinocytosis 48, we tested if macropinocytosis plays any role in RBD uptake. Several viruses 121

    utilize macropinocytosis pathway as an entry route into cells 49. Macropinocytosis is dependent on 122

    amiloride-sensitive Na+/H+ exchangers 50. Upon treatment with Amiloride, we found no alteration in 123

    the uptake of RBD, dextran and transferrin confirming that macropinocytosis does not play a role in 124

    RBD trafficking in AGS cells (Figure S2G, S2H). Together, the co-localization studies and 125

    pharmacological inhibition experiments strongly suggest that RBD uptake occurs via the CG Pathway 126

    and is inhibited by specific blockers of the CG pathway. 127

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.16.422529

  • 5

    RBD and CG uptake is blocked by endosomal acidification inhibitors – BafilomycinA1 and NH4Cl 128

    Given the relevance of acidification in both formation of CG endosomes 34,36 and in the context of viral 129

    infection 51, we focused on studying the role of acidification inhibitors on the uptake of RBD. We 130

    checked the effect of BafilomycinA1 (BafA1), a specific inhibitor of V-ATPase 52, on RBD, dextran 131

    and transferrin uptake in AGS cells. Treatment with 200nM BafA1 strongly reduced both RBD and 132

    dextran uptake (Figure 1F, 1G) and enhanced the normalized transferrin uptake (Figure 1H, 1I). This 133

    could be because BafA1 also retards the transferrin recycling from the recycling endosomes 53 and 134

    thereby increasing the net amount of transferrin internalized within cells as observed. A dose-dependent 135

    reduction in RBD and dextran uptake and an increase in transferrin uptake was seen when cells were 136

    treated with a higher concentration of BafA1 (Figure S3A, S3B (i)). We examined the effect of NH4Cl, 137

    a weak base known to alter endosomal acidification 54, on the uptake of these 3 cargoes. We observed 138

    similar results as with BafA1 (Figure S3A, S3B (i)), thus re-establishing our earlier 34 finding that uptake 139

    via the CG pathway is pH sensitive and blocking acidification results in reduced CG uptake. 140

    Towards understanding the mechanism of action for acidification inhibitors in bringing about these 141

    changes in trafficking, we assessed their effect on two parameters – numbers of endosomes (Figure S3B 142

    (ii)) and per-endosome intensity in the presence/absence of inhibitor (Figure S3B (iii)). We observed 143

    that both BafA1 and NH4Cl reduced the total number of RBD and dextran endosomes without affecting 144

    the per-endosome intensity. However, while the total number of transferrin endosomes remained 145

    unchanged, the per-endosome intensity of transferrin increased with BafA1 and NH4Cl treatment. This 146

    indicates that the reduction in RBD and dextran is likely due to a block in the entry while an increase 147

    in per-endosome transferrin intensity could be because of a block in the formation of recycling 148

    endosome carriers, as proposed earlier. 149

    We studied the effect of BafA1 on RBD uptake in cells overexpressing myc-tagged ACE2 receptor by 150

    measuring the uptake of RBD normalized to the surface ACE2 levels. We observed that BafA1 strongly 151

    affects the normalized RBD uptake (Figure S3C, S3D). HEK-293T cells, which is also permissive to 152

    Spike-pseudovirus transduction (Figure S6C), showed similar inhibition of RBD and dextran uptake, 153

    and increase in transferrin uptake with BafA1 (Figure S10A-S10D). 154

    RBD is localized to acidic compartments 155

    Internalized cargoes can be recycled along with the bulk membrane 55 or directed towards degradation 156

    with the fluid phase 56. Typically, transferrin bound to its receptor marks the early sorting and recycling 157

    endosomes and lysotracker labels the acidic degradative compartments within a cell 31. At 30 minutes 158

    of pulse with RBD, dextran and transferrin, while a small fraction of RBD (~36%) associated with 159

    transferrin, the majority of RBD (~84%) co-localized with dextran suggesting that RBD is directed 160

    predominantly towards the degradation route rather than the recycling route (Figure 1B, 1C). The 161

    lysotracker labelling showed highly acidic tubular compartments with significant co-localization with 162

    RBD. At 30 minutes of pulse with RBD, around 55% of RBD co-localized with lysotracker marked 163

    compartments. At longer time points (3 hours) of pulse with RBD, an even increased proportion of RBD 164

    (85%) associated with compartments marked by lysotracker, confirming that RBD is trafficked to acidic 165

    compartments (Figure 2A, 2B). 166

    BafilomycinA1 and NH4Cl alter the pH of acidic endosomal compartments 167

    We next focused on determining the change in endosomal pH brought about by various inhibitors within 168

    the acidic compartments populated by RBD. Cells were labelled with pH-sensitive (FITC) and pH-169

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.16.422529

  • 6

    insensitive (TMR) dextran for 2 hours and chased for 1 hour with or without inhibitors (Figure 2C, 170

    Methods). The above pulse and chase durations were chosen to allow accumulation of labelled dextran 171

    in late endosomes and lysosomal compartments (co-labelled by Lysotracker, data not shown). 172

    Additionally, since the acidification inhibitors also have inhibitory roles in the early steps of CG 173

    endocytosis as discussed in the previous section, to evaluate their effect on endosomal pH, cells were 174

    incubated with inhibitors only during the chase. While the ratio of the fluorescence of these probes is 175

    used to estimate endosomal pH by comparing the ratio with the calibration curve 57 (Figure S4A, S4B, 176

    Methods), quantifications of the endosomal intensities and the endosomal number of TMR dextran aids 177

    in understanding the effect of various drugs on late endosomal trafficking. 178

    Treatment of cells with acidification inhibitors showed an increase in endosomal pH. The average pH 179

    of the late endosomes in control cells was 5.8. The pH of these compartments increased to 6.2 and 7.1 180

    in the presence of BafA1 200nM and 400nM respectively. Incubation with NH4Cl also resulted in 181

    increasing the pH of these endosomes to 6.6 (Figure 2D, 2E, S4C). While BafA1 marginally changed 182

    the TMR intensity per endosome, NH4Cl greatly increased the TMR intensity indicating that NH4Cl 183

    also brings about the fusion of endosomes (Figure S4D). All the acidification inhibitors also reduced 184

    the numbers of endosomes (Figure S4D) and this effect was most prominent with NH4Cl wherein the 185

    endosomes were organized close to the perinuclear region (Figure S4C). The spatial pH maps show the 186

    distribution of pH of endosomes within a cell. Cells treated with BafA1 400nM and NH4Cl showed a 187

    homogenous distribution of endosomes with increased pH similar to the respective cell averages. BafA1 188

    200nM cells, on the other hand, showed heterogeneity in endosomal pH with some endosomes depicting 189

    high pH while others were closer to the average (Figure 2F). 190

    To assess the effect of BafA1 on the pH of early time point endosomes, AGS cells were labelled with 191

    FITC and TMR dextran for 20 minutes and chased for 10 minutes with or without BafA1 for the entire 192

    duration of pulse and chase (Figure S4E). While the total amount of dextran uptake is not affected 193

    significantly, the endosomal FITC intensity and the endosomal ratio of FITC/TMR, which can be 194

    considered as a proxy for endosomal pH, show a robust increase with BafA1 treatment (Figure S4F). 195

    This indicates that BafA1 also affects the endosomal pH of early time point endosomes. 196

    Chloroquine treatment does not affect RBD uptake and minimally alters endosomal pH 197

    Chloroquine, a diprotic weak base, is expected to accumulate in acidic compartments and neutralize 198

    lysosomal pH 58. While, mounting evidence shows that Chloroquine and its analogs can inhibit the 199

    infection by several viruses such as Ebola, Dengue, Chikungunya, HIV, etc 59, many studies point 200

    towards differences between the mode of action of Chloroquine and acidification inhibitors – BafA1 201

    and NH4Cl 60,61. We, therefore, tested the effect of Chloroquine on RBD, dextran and transferrin uptake 202

    to verify if it behaves like BafA1. We found that upon treatment with Chloroquine, the uptake of neither 203

    RBD nor transferrin was altered significantly (Figure S5A, S5B). Dextran uptake was marginally higher 204

    upon treatment with Chloroquine (Figure S5B). 205

    The effect of Chloroquine in changing the endosomal pH of late endosomes using FITC/TMR ratio as 206

    a proxy for endosomal pH was assessed. At different concentrations of Chloroquine tested, the 207

    endosomal pH was only minimally increased (Figure S5C, S5D). We also observed that both FITC and 208

    TMR endosomal intensities increased with the concentration of Chloroquine. To confirm our results, 209

    we used another method to estimate endosomal pH. FITC has a pH-sensitive (488nm) and a pH-210

    insensitive excitation (450nm) 54. We used the 488/458 excitation ratio of FITC dextran as a readout of 211

    pH and found that this ratio also showed only a small albeit significant increase with Chloroquine when 212

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.16.422529

  • 7

    compared to control cells, unlike the increase brought about by NH4Cl (Figure S5E, S5F). This 213

    observation could explain the lack of an endocytic effect on RBD, dextran or transferrin uptake upon 214

    treatment with Chloroquine. 215

    Designing Spike-pseudovirus transduction assay to specifically address the effects of inhibitors on viral 216

    entry 217

    To ascertain that the observations made using the RBD, as a proxy for viral entry, are valid in the context 218

    of a viral entity decorated by the SARS-CoV2 Spike protein itself, we generated SARS-CoV2 Spike-219

    pseudotyped lentiviral particles (Spike-pseudovirus), following a previously established methodology 220 62. mCherry fluorescent protein expression was used as a reporter for assessing viral infection (Figure 221

    S6A, Methods). The expression of the Spike protein in the pseudovirus particles was verified by a 222

    western blot using the antibody against the C-terminal Strep-tag of Spike, which revealed bands 223

    corresponding to both the S2 fragment as well as the full-length protein (Figure S6B). The infection 224

    specificity of the pseudovirus was validated by infection of human (HEK-293T) versus mouse (NIH-225

    3T3) cells, where the latter showed lower infectivity, consistent with a lack of a bonafide hACE2 226

    receptor to bind the Spike protein (Figure S6C). Independently, a competition experiment was 227

    conducted to check the effect of excess soluble RBD on transduction of Spike-pseudovirus in HEK-228

    293T (Methods). The transduction efficiency was reduced in the presence of soluble RBD, indicating 229

    that the Spike-pseudovirus competes for the same binding sites as RBD (Figure S6D). However, the 230

    inhibition was not complete, possibly since even high concentration of free RBD in the solution cannot 231

    compete with the high effective concentrations on Spike-pseudoviruses, augmented even more by their 232

    trimeric configuration that facilitates multi-valent interactions 63. 233

    Since our experiments were aimed at understanding the entry mechanism of Spike-pseudovirus, we 234

    designed the transduction assays with shorter pseudovirus incubation time and followed the infection 235

    efficiency by tracing reporter gene expression at a later time point. We characterized the transduction 236

    efficiency of the pseudovirus as a function of its MOI and time of incubation to obtain an optimum MOI 237

    and incubation time (Figure S6E). Transduction efficiency measured across the tested regime suggested 238

    4 or 8 hours of incubation at 0.5 MOI to be optimal to achieve at least >1000 positive cells (per well of 239

    a 96-well assay plate) with reasonably low viral load and incubation time. 240

    Spike-pseudovirus transduction is reduced by endosomal acidification inhibitors and Chloroquine 241

    If the pseudovirus expressing the full-length spike mimicked the same trafficking pathway for entry as 242

    RBD, we reasoned that the transduction efficiency would be reduced upon treatment with inhibitors 243

    affecting RBD uptake. SARS-CoV2 Spike-pseudovirus transduction efficiency has been reported to be 244

    reduced upon treatment with NH4Cl, BafA1 and Chloroquine 24,64. However, we wanted to specifically 245

    explore the actions of these inhibitors at the initial stages of infection. Therefore, to address this, we 246

    pre-treated cells with drugs for an hour followed by the addition of Spike-pseudovirus in the presence 247

    of the drug for 2, 4 or 8 hours. Both virus and drug were removed thereafter, and cells were incubated 248

    with fresh media either in the absence or presence of a minimal concentration of the drug as indicated 249

    (Figure 3A, Methods). This design was chosen to reduce long-term toxicity of the inhibitors to the cells 250

    and minimize any secondary effects on the translational processes of the reporter gene post entry. 251

    Infection, or transduction efficiency, is reported as the normalized percentage of transduction compared 252

    to corresponding control and cell viability is measured in terms of nuclei number normalized to control. 253

    We tested the effect of NH4Cl and BafA1 on the Spike-pseudovirus transduction assay in AGS cells 254

    upon treatment with the 20mM NH4Cl or 50nM BafA1. At the end of designated time points, the 255

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.16.422529

  • 8

    pseudovirus containing media along with the NH4Cl and BafA1 was removed and replenished with 256

    fresh media alone. We observed a significant reduction of Spike-pseudovirus transduction with NH4Cl 257

    and BafA1 compared to the corresponding controls with no significant difference in cell viability at the 258

    end of both 4 and 8 hours (Figure 3B-3E). NH4Cl also shows robust reduction even upon 2 hours of 259

    incubation (Figure 3E). HEK-293T cells also exhibited a similar inhibition of transduction upon 260

    treatment with 20mM NH4Cl or 50nM BafA1 (Figure S10 E-G). 261

    Although Chloroquine did not alter RBD uptake or increase the pH of the endocytic compartments 262

    significantly, we observed a marked reduction of viral transduction with 50µM Chloroquine treatment 263

    in AGS cells (Figure 3D, 3E) and 10µM Chloroquine treatment in HEK-293T cells (Figure S10E-G), 264

    where the drug was removed along with the Spike-pseudovirus at the end of 8 hours of incubation. 265

    Upon testing whether long term incubation of Chloroquine (as used in this assay), results in changes in 266

    endosomal pH or RBD uptake, we observed no alterations in the assessed phenotypes in AGS cells 267

    (Figure S5G, S5H). This suggests a distinct pH-independent mechanism of intervention by 268

    Chloroquine, functioning at the initial stages of infection. 269

    RBD uptake is reduced upon treatment with AN96 and ML141, albeit to a lesser extent compared to 270

    the effect of BafA1 and NH4Cl. Therefore, we assessed the effect of AN96 and ML141 on Spike-271

    pseudovirus transduction in AGS cells, using the 2 hours and 8 hours format of the assay, respectively. 272

    The AN96 concentration was reduced to a non-toxic level of 1µM after removal of the virus. With this 273

    experimental design, 5 different concentrations of AN-96 were tested, and we observed no reduction in 274

    normalized percentage transduction even at the highest concentration of 25µM (Fig. 3F and G), with 275

    no compromise on cell viability. This was consistent with the observation that RBD is rerouted and 276

    associates more with transferrin. In case of ML141, we treated the cells with 5µM of the drug and 277

    observed no difference in normalized percentage transduction compared to the control (Figure S6F, 278

    S6G). Partial inhibition of uptake may not strongly manifest in our pseudovirus assay, as the read-out 279

    is all or none and is not sensitive to the number of virus particles entering the cells. Our findings suggest 280

    that inhibitors that affect both RBD uptake and neutralize acidic endosomes could be one of the 281

    strategies used to impede Spike-pseudovirus transduction. 282

    Identifying FDA-approved drugs functioning similar to BafA1 and NH4Cl 283

    Armed with the knowledge on the mode of action of acidification inhibitors in reducing the uptake of 284

    RBD, increasing the pH of endosomes and abrogating the infection of Spike-pseuodovirus, we screened 285

    a small subset of FDA-approved drugs with the potential to alter the pH of endosomes (Figure 4A). We 286

    selected a panel of 6 drugs which includes those acting on Na+/K+ ATPase (Omeprazole, 287

    Esomeprazole, Pantoprazole, SCH-28080, Lansoprazole) and a protonophore that disrupts proton 288

    gradient (Niclosamide). We developed a quantitative high throughput screening pipeline for testing 289

    these drugs in both endocytic assay as well as pH estimation assay in AGS cells. The screen was carried 290

    out at a concentration of 10µM for all drugs. 291

    Of the 6 drugs tested in the endocytosis assay, Niclosamide showed the strongest effect on the uptake 292

    of the 3 probes (RBD, dextran and transferrin) similar to what we observed for acidification inhibitors. 293

    Niclosamide treated cells showed reduced RBD and dextran uptake and increased transferrin uptake 294

    (Figure 4B, 4C). It is interesting to note that while the other proton pump inhibitors had minimal effects 295

    on RBD or dextran uptake at the concentration tested, Omeprazole and Pantoprazole showed a 296

    significant increase in transferrin uptake (Figure 4C, S7A). This suggests that these two drugs could 297

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

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  • 9

    specifically act on the transferrin containing endosomes and not in the compartments of relevance for 298

    RBD and dextran uptake, while Niclosamide inhibits the RBD and dextran uptake. 299

    Of the 6 drugs tested in the late endosomal pH estimation assay, Niclosamide also showed the strongest 300

    neutralization effect on the pH of acidic endosomes (Figure 4E) by increasing the endosomal ratio of 301

    FITC/TMR (Figure S7C). The other drugs had minimal effects on the pH of late endosomes at the 302

    concentration tested (Figure 5E, S7B). The spatial pH maps of Niclosamide treated cells show an 303

    increase in pH in the majority of endosomes within the cell (Figure 5D). Niclosamide increased the 304

    FITC endosomal intensity and reduced the numbers of endosomes (Figure S7C) similar to the effect of 305

    BafA1 on these endosomal trafficking parameters. 306

    Omeprazole and other proton pump inhibitors are prodrugs which are used for treating Gastro-307

    esophageal reflux disease (GERD) 65. They are activated by low pH, bind covalently to H+/K+ ATPase 308

    and inhibit the enzymatic function 66. We tested the hypothesis if these drugs could also similarly block 309

    the proton pumps in the late endosomes and thus increase the endosomal pH 67,68. Earlier studies have 310

    indicated that Omeprazole 69, Lansoprazole 70, and Pantoprazole 71, neutralize the endosomal pH only 311

    when used at very high concentrations (> 1mM) in EMT-6 and MCF-7 cells. However, the plasma 312

    concentration of these proton pump inhibitors varies between 1–23µM 65. Thus, at least in the 313

    concentration range of relevance, we find no effect of these drugs on the acidification of endosomes 314

    and the uptake of RBD. 315

    Niclosamide functions as an acidification and entry inhibitor 316

    Niclosamide is an anti-helminthic FDA-approved drug and has been in use since the 1960s (Ditzel, 317

    1967). Many recent studies show that Niclosamide has broader clinical applications and has also been 318

    identified as an antiviral against SARS-CoV, human Rhinovirus, Influenza viral, Dengue virus 72,73. As 319

    Niclosamide emerged as a potential drug candidate in both the RBD endocytosis as well as endosomal 320

    pH neutralization screens, we investigated the dose-dependent role of Niclosamide in reducing RBD 321

    uptake, neutralizing endosomal pH and inhibiting Spike-pseudovirus infection. We found that 322

    Niclosamide reduced both RBD and dextran uptake, as well as increase transferrin uptake in a dose-323

    dependent manner (1–25µM) (Figure 5A, S8A). We observed Niclosamide’s effect on RBD 324

    endocytosis even at concentrations as low as 1µM. On analyzing the effect of Niclosamide on 325

    endosomal numbers and intensity, we found that Niclosamide increased the endosomal intensity of 326

    transferrin endosomes and reduced the number of RBD and dextran endosomes (Figure S8B). These 327

    effects are remarkably similar to the effects observed with acidification inhibitors – BafA1 and NH4Cl. 328

    We also confirmed the inhibitory effect of Niclosamide on RBD and dextran uptake in another cell line 329

    – HEK-293T (Figure S10A-D), and on normalized RBD uptake in AGS cells overexpressing ACE2 330

    (Figure S3C, S3D). 331

    Further, we also observed a dose-dependent effect of Niclosamide on neutralizing the pH of late 332

    endosomes, with neutralization effects seen even at 2.5µM (Figure 5B, 5C). The dose-response effect 333

    is seen on the ratio of endosomal FITC/TMR as well as other endosomal trafficking parameters - FITC 334

    and TMR endosomal intensities and numbers of endosomes (Figure S8C). The spatial pH maps of cells 335

    also show a gradual shift of endosomal pH from acidic to neutral pH with different doses of Niclosamide 336

    (Figure 5B), especially at 2.5µM wherein some endosomes within the cell are still acidic while some 337

    others are neutralized. Towards evaluating the effect of Niclosamide on the pH of early time point 338

    endosomes, AGS cells were labelled with FITC and TMR dextran for 20 minutes and chased for 10 339

    minutes with or without Niclosamide for the entire duration of pulse and chase (Figure S4E). Unlike 340

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

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  • 10

    BafA1, while Niclosamide reduced the net uptake of dextran, similar to BafA1, Niclosamide also 341

    increased the endosomal FITC intensity and endosomal FITC/TMR ratio of early time point (30 342

    minutes) endosomes (Figure S4F), indicating that Niclosamide neutralizes the pH of these endosomes 343

    as well. 344

    We assessed the effect of different concentrations (0.1-10 µM) of Niclosamide on Spike-pseudovirus 345

    entry in AGS cells, using the experimental strategy designed to assess virus entry as described before. 346

    At the end of 4 and 8 hours of viral incubation, the pseudovirus containing media along with the 347

    Niclosamide was removed and all treatments were replenished with media containing a reduced 348

    concentration of 0.1µM Niclosamide. We observed a strong dose-dependent reduction of transduction 349

    efficiency as a function of increasing Niclosamide concentration at both viral incubation durations with 350

    negligible toxicity (Figure 5D, S9Ai and Aii). IC50 of ~1.27µM was estimated on fitting a sigmoidal 351

    function to the dataset obtained for 8 hours of viral incubation (Figure 5E, Methods). Together, all three 352

    assays conclude that Niclosamide can act as acidification and entry inhibitor. 353

    Enhancing the inhibition of infectivity: A combination strategy with Niclosamide and 354

    Hydroxychloroquine 355

    A combinatorial approach of drugs with varying mechanisms of inhibition works as an effective therapy 356

    to combat infection 74. Given that Niclosamide exhibits a short half-life 75, has poor bio-availability 357

    (~10 %) 76 and our observations indicate moderate IC50 for inhibition of Spike-pseudovirus transduction, 358

    we tested if the action of Niclosamide can be enhanced in the presence of another FDA-approved drug 359

    known to be effective against SARS-CoV2 infection. Since published reports and commonly practiced 360

    treatments against SARS-CoV2 infection employ Hydroxychloroquine (HCQ), a less toxic variant of 361

    Chloroquine 77, we tested the effect of HCQ on altering late endosomal pH and Spike-pseudovirus 362

    transduction assay. Like Chloroquine, cells treated with 50µM HCQ also minimally altered the late 363

    endosomal pH (Figure S9B, S9C). However, we observed the pseudovirus transduction to be markedly 364

    reduced at HCQ concentrations of 50µM and 25µM (Figure S9D, S9E) and only modestly reduced at 365

    the concentrations of 10µM or lower in a dose-dependent manner (See the first box plot in Figures S9 366

    Fi, Fii and Fiii). To assess the synergistic effect of the two drugs, we chose a concentration range with 367

    the maximum concentrations of 10µM HCQ and 5µM Niclosamide. The 2-dimensional dose-response 368

    map shown in Figure 5G summarizes the effect of the two drugs on transduction. We observed an 369

    augmented reduction in infection when HCQ was used at a concentration of 10µM along with varying 370

    concentrations of Niclosamide compared to where HCQ was used at 0, 2 and 5µM (Figure S9Fi, Fii 371

    and Fiii). These results indicate an additive effect on inhibition of pseudovirus transduction when 372

    effective concentrations of HCQ is added along with effective concentrations of Niclosamide (Figure 373

    5F, 5G). Thus, Niclosamide could potentially enhance the efficacy of the plethora of treatments 374

    currently being used to combat SARS-CoV2 infection. 375

    Discussion: 376

    Understanding the molecular mechanisms of viral entry into target cells is critical to design effective 377

    treatments and prevention strategies against infection. Employing various methodologies, we report for 378

    the first time that fluorescently labelled RBD of SARS-CoV2 enters cells through a pH-dependent CG 379

    pathway. High-resolution quantitative imaging approaches enabled us to detect the localization of RBD 380

    to acidic compartments. Endosomal acidification inhibitors that affect the uptake of CG cargo also 381

    inhibit RBD uptake. Complementing our observations with RBD, we show that infection by Spike-382

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

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  • 11

    pseudovirus is also dependent on endosomal acidification. Further, by employing a targeted drug screen, 383

    we have identified Niclosamide as a potential inhibitor against SARS-CoV2 entry (Figure 6). 384

    The choice of viral entry into host cells is influenced by cell surface interacting partners and co-factors 385 11,25. Although ACE2 has been identified as the receptor for SARS-CoV2, other receptors are being 386

    uncovered. These include Neuropilin 12,13, CD147 14, Heparan Sulphate proteoglycans 15 and HDL 387

    scavenger receptors 16. Additionally, the highly glycosylated nature of Spike protein could also confer 388

    the ability to interact with yet unidentified receptors. These virus-receptor interactions could potentially 389

    dictate the endocytic route employed by the virus. This is exemplified by our observation that although 390

    RBD uptake is reduced upon blocking the CG pathway, residual RBD re-routes towards the CME and 391

    enables pseudovirus infection. Re-routing could presumably be due to binding to different receptors 392

    that could follow alternative internalization routes. Whether the Spike-pseudovirus follows routes of 393

    entry like RBD, can be addressed with tractable pseudoviruses or synthetic virus-like particles. 394

    However, recent genome wide screens 38,78 indicating the importance of cholesterol homeostasis in 395

    SARS-CoV2 infection are consistent with a cholesterol senstitive CG endocytic route 79 in entry. 396

    Knockouts of genes affecting cholesterol biosynthesis (SCAP, MBTSP1, MBTSP2) not only reduced 397

    infection of native SARS-CoV2 but also of Spike-pseuodviruses indicating that cellular cholesterol is 398

    necessary for efficient Spike mediated entry of SARS-CoV2 78. 399

    Known inhibitors of endosomal acidification, BafilomycinA1 and NH4Cl, play an important role in 400

    neutralizing acidic lysosomes and thus subverting viral membrane fusion and entry of several viruses 401 11,24–27. Here, we report that these inhibitors also play a more upstream role by inhibiting the endocytosis 402

    of RBD itself. Both these treatments inhibited the uptake of CG cargo and RBD, reduced Spike-403

    pseudovirus infection and drastically elevated endosomal pH. It is interesting to note that the inhibition 404

    of acidification in addition to dramatically reducing CG uptake did not cause re-trafficking of RBD 405

    through another endocytic pathway, as was observed for other CG inhibitors. This suggests that the 406

    acidification inhibitors could negatively influence the RBD-receptor interactions at the cell surface 407

    along with further ramifications of blocking the CG pathway. 408

    These observations encouraged us to screen a subset of FDA-approved compounds known to affect 409

    endosomal acidification: proton-pump inhibitors (Omeprazole, Lansoprazole, Pantoprazole, 410

    Esomeprazole, SCH-28080), and protonophore (Niclosamide). Of all the 6 compounds tested only 411

    Niclosamide inhibited CG cargo and RBD uptake, elevated endosomal pH and concomitantly inhibited 412

    Spike-pseudovirus infection, all in a dose-dependent manner with an IC50 of 1.27 µM in AGS cells. 413

    Among several mechanisms of action 75, Niclosamide disrupts proton gradient across mitochondrial 80 414

    and endosomal 72 membranes. The elevated endosomal pH brought about by Niclosamide was shown 415

    to inhibit human rhinovirus infection 72. Additionally, Niclosamide has been identified as an anti-viral 416

    agent against SARS 81, Dengue 73, MERS 82 and more recently proposed for SARS-CoV2 (with IC50 of 417

    0.28 µM in Vero cells) 83, although the mechanism of action remained unknown. In contrast, the proton 418

    pump inhibitors used in our study failed to interfere with RBD uptake. This could be because they 419

    remained inactive 65 or the concentrations tested predominantly affect H+/K+ ATPases, while mM 420

    concentrations are required to inhibit V-ATPases 67. Along these lines, studies show that proton pump 421

    inhibitors inhibit Ebola-pseudovirus 84, SARS-CoV and SARS-CoV2 85 infection only when used 422

    beyond achievable plasma concentrations 65. 423

    Surprisingly, Chloroquine did not affect RBD uptake and only marginally raised the endosomal pH. 424

    However, it caused a strong inhibition of Spike-pseudovirus infection. This strongly suggests that 425

    Chloroquine could be operating in the initial steps of viral infection but post endocytosis 64, as observed 426

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

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  • 12

    with RBD uptake. Chloroquine is likely to function in many pH-independent ways to inhibit SARS-427

    CoV2 infections, distinct from Niclosamide. For example, by altering terminal glycosylation of ACE2 428 86; via its activity as a zinc ionophore affecting ACE2 activation 87,88; by interacting with ER resident 429

    Sigma receptors that initiates cell stress response 89; by its ability to strongly bind a viral protease 430

    essential for Spike activation 90. At this time, the exact mechanism(s) by which Chloroquine inhibits 431

    SARS-CoV2 entry remains unclear. 432

    In conclusion, our study reports the high capacity CG pathway as a potential endocytic route for SARS-433

    CoV2. We further show that endosomal acidification is critical for SARS-CoV2 entry and infection and 434

    can be a promising therapeutic target as observed by the results seen with Niclosamide, BafilomycinA1 435

    and NH4Cl. This study also paves way for large-scale screens to repurpose FDA-approved drugs as 436

    acidification inhibitors and scrutinize for more Niclosamide-like drugs that might have better 437

    bioavailability or can be used in combination with other antiviral drugs. Moreover, the methods 438

    described in our study can effectively be extended and better represented with clinical isolates of viruses 439

    to assess their infective journey in primary cells that represent the more natural hosts for infection. 440

    Materials and Methods: 441

    Cell lines, constructs, and antibodies: See supplementary methods for more details 442

    Chemicals and reagents: 443

    Niclosamide and AN96 were chemically synthesized and proton pump inhibitors, Esomeprazole and 444

    Pantoprazole, were extracted from commercially available tablets as detailed in the Supplementary 445

    Methods. The other proton pump inhibitors, Lansoprazole and SCH-28080, were obtained from the 446

    LOPAC®1280 library, and Omeprazole was procured from Sigma (O104). 447

    Endocytosis assays: 448

    AGS or HEK-293T cells were plated in 35mm coverslip bottom dishes and processed after 48 hours at 449

    60-70% confluency. Cells were washed twice with HEPES buffer (wash and imaging buffer 450

    composition: 150mM NaCl, 20mM HEPES, 5mM KCl, 1mM CaCl2, 1mM MgCl2, 2mg/ml Glucose, 451

    pH 7.5) at 37C. Endocytosis was monitored using fluorescently labelled RBD (Alexa/Atto 488, 452

    10g/ml), 10kDa TMR-dextran (1mg/ml) and/or Iron-loaded Transferrin (10g/ml, Alexa 647) in 453

    serum-free medium for indicated time points at 37C. Endocytosis was stopped using ice-cold wash 454

    buffer and cells were subsequently fixed with 2.5% paraformaldehyde (PFA) for 20 minutes at room 455

    temperature (RT). Cells were then washed and imaged. For inhibitor experiments, cells were pre-treated 456

    with various inhibitors (AN96 25M, ML141 50M, Amiloride 1mM, BafA1 200nM or 400nM, NH4Cl 457

    30mM) and respective controls in serum-free medium for 30 minutes at 37C and inhibitors were 458

    maintained during endocytic assays. 459

    To measure normalized transferrin or normalized RBD uptake (Figures 1H-1I, S3C-S3D), cell surface-460

    bound probes after the endocytic pulse with transferrin or RBD were stripped using two washes with 461

    ice-cold ascorbate buffer (160mM sodium ascorbate, 40mM ascorbic acid, 1mM MgCl2, 1mM CaCl2, 462

    pH 4.5), followed by three washes with ice-cold wash buffer at 4 °C. Cells were then fixed with ice-463

    cold 2.5% PFA for 5 mins at 4 °C and 15 minutes at RT. Transferrin receptor (TfR) was labelled by 464

    incubating cells with fluorescently labelled anti-hTfR (OKT-9) for 2 hours at RT. To label surface 465

    ACE2, fixed cells were blocked with 10mg/ml bovine serum albumin (30 minutes) followed by 466

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  • 13

    incubation with anti-myc primary antibody (1 hour) and secondary antibody (45 minutes) in blocking 467

    buffer at RT. Cells were then washed and imaged. 468

    pH estimation assays: 469

    For estimating the pH of late endosomes, cells were pulsed with pH-sensitive 10kDa FITC-dextran 470

    (1mg/ml) and pH-insensitive 10kDa TMR-dextran (1mg/ml) for 2 hours in serum-free media, chased 471

    for 1 hour in the presence of inhibitors or control and imaged live. The above pulse and chase times 472

    were chosen to allow the accumulation of labelled dextran in acidic late endosomal and lysosomal 473

    compartments (co-labelled with Lysotracker, data not shown). To estimate the endosomal pH, the ratio 474

    of FITC to TMR fluorescence was computed and compared to a pH calibration curve (Figures S4A-475

    S4B) which was generated by equalizing the endosomal pH to that of an external buffer. After the pulse 476

    with FITC and TMR-dextran and chase, cells were incubated with 5µg/ml nigericin containing buffers 477

    of different pH for 10 minutes and imaged to evaluate FITC/TMR ratios for each pH. 478

    For estimating the pH of late endosomes using the 488/458 excitation ratio of FITC-dextran (Figures 479

    S5E-S5F), cells were pulsed with FITC-dextran at 1mg/ml for 2 hours, followed by chase in the 480

    presence or absence of inhibitors and imaged live. 481

    For estimating the FITC/TMR ratio of early endosomes (Figures S4E-S4F), cells were incubated with 482

    pH-sensitive 10kDa FITC-dextran (1mg/ml) and pH-insensitive 10kDa TMR-dextran (1mg/ml) for 20 483

    minutes, chased for 10 minutes and imaged live. Throughout the pulse and chase duration, the cells 484

    were incubated in serum-free media with control (0.2%DMSO) or BafA1 400nM or Niclosamide 485

    10µM. 486

    Spike-pseudovirus transduction assays: 487

    AGS/HEK-293T cells were plated in optical bottom 96-well plates. 36 hours post-plating, when cell 488

    numbers were ~4000, transduction was carried out at indicated MOIs. For inhibitor treatment, cells 489

    were pre-incubated with indicated concentrations of NH4Cl/ BafA1/ CQ/ Niclosamide/ HCQ/ AN96/ 490

    ML141, for 1 hour. This was followed by addition of the Spike-pseudoviruses in presence or absence 491

    of the inhibitors. At the end of 2/4/8hours, media containing pseudoviruses and inhibitors was removed, 492

    and cells were washed once with drug-free media. This was followed by addition of media with or 493

    without inhibitor: NH4Cl, BafA1 and CQ were removed from the media; Niclosamide, AN96 and HCQ 494

    were maintained at a low concentration of 100nM, 1µM and 500nM respectively. This was done to 495

    assess the effects of the inhibitors at the initial stages of inhibition, minimize long-term toxicity to the 496

    cells as well as to avoid effects on the translational processes of the reporter gene post entry. After 60 497

    hours, cells were fixed, nuclei were labelled with Hoescht and assessed for transduction efficiency based 498

    on mCherry reporter expression. In the case of HEK-293T cells (Figure S10G), MTT cell viability assay 499

    was performed to check toxicity (assay described in Supplementary Methods). 500

    Imaging and Analysis: 501

    a. Endocytic and pH estimation assays 502

    For 35mm dish-based endocytic experiments, fixed samples were imaged using confocal microscopy 503

    (Olympus FV3000, 20X/0.85NA objective) to image RBD, dextran and transferrin endosomes with Z 504

    sections of 1µm. Maximum intensity projected images were used for further analysis. Cell ROIs were 505

    drawn and features such as cell mean intensity in each channel was extracted. 506

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

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  • 14

    For high-throughput endocytic and pH estimation experiments, automated imaging (Spinning disc, 507

    Phenix Perkin Elmer, 40XW/1.1NA objective) was used to image nucleus along with RBD, dextran 508

    and transferrin (for endocytosis) or FITC and TMR dextran (for pH) with Z sections of 1µm each. For 509

    both assays, cell profiler based pipeline was used to segment cells, nucleus and endosomes and extract 510

    features as described in supplementary methods. For pH calibration, the mean of the endosomal ratio 511

    distributions at different extracellular pH was fit to a sigmoidal equation. For both assays, custom 512

    MATLAB routines were used to estimate the endosomal intensities, the number of endosomes and cell 513

    mean intensities. In addition, for pH assays, endosomal ratio (FITC/TMR) and endosomal pH (using 514

    the calibration curve) for each endosome was computed. As the endosomal intensity distribution within 515

    cells is a heavy right-tailed distribution, median endosomal intensity for each probe for each cell was 516

    estimated. The distributions of cell mean intensity/endosomal intensities/numbers of endosomes per 517

    cell per treatment (for endocytosis) and endosomal intensities/ratio/pH per cell per treatment (for pH) 518

    is represented in each quantification. 519

    For 488/458 endosomal ratio estimation experiments, live imaging was done using confocal microscopy 520

    (Zeiss LSM 780, 40X/1.4NA objective). Excitation lasers 488nm, 458nm were used and emission was 521

    detected using a spectral detector (490nm-560nm). Images were processed as described above to 522

    estimate endosomal intensities and endosomal ratios per cell. 523

    b. Colocalization analysis - Confocal microscopy (Olympus FV3000, 60X/1.42NA objective) with Z 524

    sections of 0.4µm each was employed to image cells across all channels. A MATLAB routine was 525

    written to extract colocalization indices. For each cell, endosomes in each channel were segmented 526

    based on threshold values. The segmentation in each channel was made finer using morphological 527

    operations (dilation followed by erosion). Segmented endosomes were considered for colocalization 528

    analysis. Manders’ coefficients and Pearson’s correlation coefficients were computed as described 529

    before 91. 530

    c. Pseudovirus transduction assays - Automated imaging (Widefield, Phenix, 10X/0.3NA objective) of 531

    96 well assay plates was used to image nucleus as well as mCherry positive cells. A cell profiler based 532

    pipeline was used to segment nucleus and extract features, as described in supplementary methods. 533

    Approximately 50,000 nuclei (cells) were scored for each treatment. A MATLAB routine was written 534

    to estimate the % transduction. Mean intensities of the segmented nucleus in the nuclei channel and the 535

    mCherry channel for each nucleus across all fields were extracted. Each assay plate included “No-536

    Virus” negative control. This control was used to estimate the background intensities of the mCherry 537

    channel within each segmented nucleus. The median of this distribution was considered the background. 538

    All nuclei with mCherry intensities of at least 1.8 - 2.2 times (empirically determined) the intensity of 539

    the background were considered positive. For each field, the fraction of positive nuclei to the total 540

    number of nuclei was determined. The mean of % transduction across all fields for each treatment was 541

    calculated. The % transduction was normalized to that of the control and is represented in all the 542

    quantifications. The total number of nuclei for each treatment is also represented to understand the 543

    effect of the toxicity of drugs. 544

    Statistical methods and hypothesis testing: 545

    All statistical tests between control and treatment were performed in MATLAB using Wilcoxon rank-546

    sum test and the p-value of the hypothesis testing and the number of repeats is indicated in figure 547

    legends. In the entire manuscript, ***, **, * and ns indicate p-value of Wilcoxon rank-sum test < 0.001, 548

  • 15

    magnitude of differences as well as the sample size. For large sample size, as in case of our high 550

    throughput experiments, the impact of random error in measurement will be reduced and the larger 551

    magnitude of difference between the control and treatment will be associated with a much smaller p-552

    value. 553

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    https://doi.org/10.1101/2020.12.16.422529

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    Acknowledgements: 759

    We thank Nevan Krogan (UCSF, USA) for Spike expression construct, Florian Krammer (Mt. Sinai, 760

    USA) for secreted RBD expression construct, Biocon, Ltd (India) and Raghavan Varadharajan (IISc, 761

    India) for providing purified RBD for initial experiments, Minhaj Sirajuddin (inSTEM, India) for 762

    mCherry expression plasmid, Vinoth Kumar (inSTEM, India) for discussions on Spike-pseudovirus 763

    characterization and Mylan Laboratories (India) for providing HCQ. We thank the Central Imaging and 764

    Flow Cytometry Facility (CIFF) and the Screening Facility at NCBS, for imaging and high content 765

    screening. We sincerely thank Abrar Bhat (for help with RBD purification), Chandrima Patra (for help 766

    with imaging), Greeshma Pradeep (for help with microscopes), Sarayu Beri (for help with cell culture) 767

    and all members of SM lab for exciting discussions on this project. We acknowledge the technical, 768

    administrative and hospitality staff at NCBS for tremendous help especially during the national 769

    lockdown. We acknowledge NCBS-TIFR graduate fellowship (for CP and PS), UGC graduate 770

    fellowship (for RG) and NCBS postdoctoral fellowship (for SJ). TSvZ acknowledges EMBO 771

    postdoctoral fellowship (ALTF 1519-2013) and NCBS Campus fellowship; AC thanks India Alliance 772

    DBT – Wellcome Trust Early career fellowship (IA/E/15/1/502339); SM acknowledges J.C. Bose 773

    Fellowship from DST, Government of India, and India Alliance DBT – Wellcome Trust Margdarshi 774

    fellowship (IA/M/15/1/502018). CP, PS and SJ received support from Margadarshi fellowship grant 775

    (IA/M/15/1/502018). 776

    Author Contributions: 777

    CP, RG, PS, AG, VS and SM conceived the study. CP, RG, PS, SJ, AG, VS and SM designed the 778

    experiments. Endocytosis and pH assays were done by RG and CP, Spike-pseudovirus assays were 779

    done by PS and SJ. CP, TSvZ and DS acquired and analyzed data. CP, RG, PS, SJ, TSvZ, DS, AG, VS 780

    and SM interpreted the data. RG, NS, SML, DS and CP conducted high-throughput experiments. AC 781

    and PS prepared Spike-pseudovirus. VKN and SBB synthesized Niclosamide. RA, AHN, PPS and RV 782

    synthesized AN96. TPP and PV extracted Esomeprazole and Pantoprazole. CP, SD, BM and PS purified 783

    and labelled RBD. CP, RG, PS, SJ, TSvZ and SM wrote the manuscript with comments from AC, AG 784

    and VS. 785

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.16.422529doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.16.422529

  • 22

    Main Figure Legends: 1

    Figure 1: RBD uptake is sensitive to CG Pathway inhibitors in AGS cells 2

    A: Schematic describing endocytic pathways at the plasma membrane with specific cargoes for each 3

    endocytic pathway: transferrin (CME Cargo) and 10kDa dextran (CG Cargo). AN96, ML141 and 4

    BafA1 specifically affect the uptake of CG cargoes. 5

    B, C: AGS cells were pulsed with RBD, dextran and transferrin for 30 minutes and imaged at high 6

    resolution after fixation. Images are shown in B and quantification of Manders’ co-occurrence 7

    coefficient is shown in C. This compares the fraction of RBD endosomal intensity with transferrin or 8

    dextran (p-value < e-06), transferrin endosomal intensity with dextran or RBD (p-value < e-05) and 9

    dextran endosomal intensity with transferrin or RBD (p-value = 0.18). RBD is more co-localized to 10

    dextran endosomes. Number of cells = 10. White arrow represents endosomes containing RBD, dextran 11

    and transferrin. Yellow arrow represents endosomes with RBD and dextran without transferrin. Dashed 12

    white line in B represents the approximate cell boundary. 13

    D, E: AGS cells were pretreated with Control (0.6% DMSO) or AN96 25µM for 30 minutes and pulsed 14

    with RBD, dextran and transferrin for 30 minutes with or without the inhibitor. Treatment with AN96 15

    reduces RBD (p-value < e-19) and dextran (p < e-44) uptake while minimally alters transferrin uptake 16

    (p = 0.02). Images are shown in D and quantification in E. Numbers of cells > 100 for each treatment. 17

    F, G: AGS cells were treated with Control (0.2% DMSO) or BafA1 200nM for 30 minutes and pulsed 18

    with RBD and dextran for 30 minutes with or without the inhibitor. Treatment with BafA1 reduces 19

    RBD (p-value < e-33) and dextran (p-value < e-18) uptake. Images are shown in F and quantification 20

    in G. Numbers of cells > 100 for each treatment. 21

    H, I: AGS cells were treated with Control (0.2% DMSO) or BafA1 200nM for 30 minutes and pulsed 22

    with RBD and transferrin for 30 minutes with or without the inhibitor. The surface transferrin receptor 23

    (TfR) was labelled after fixation. Treatment with BafA1 reduces RBD uptake (p-value < e-27) and 24

    increased normalized transferrin uptake (p-value < e-03). Images are shown in H and quantification in 25

    I. Numbers of cells > 80 for each treatment. 26

    Data (E, G, I) is represented as a scatter with box plot. Black dots represent per-cell data points. Box 27

    plot represents the distribution (25% to 75% percentile) with the red line indicating the median and red 28

    dot indicating the mean of the distribution. Whiskers represent distribution up to 1.5 times interquartile 29

    range and + indicates outliers beyond the whiskers. In the entire manuscript, ***, **, * and ns indicate 30

    p-value of Wilcoxon rank-sum test < 0.001,

  • 23

    more Lysotracker positive compartments have RBD (p-value < e-06). Each condition has >12 cells. 39

    Dashed white line in A represents approximate cell boundary. 40

    C: Schematic describing the experimental protocol for estimating the pH of endosomes by ratiometric 41

    measurements using pH-sensitive (FITC) and pH-insensitive (TMR) dextran. 42

    D-F: AGS cells were pulsed with FITC and TMR dextran for 2 hours, chased for 1 hour with BafA1 43

    200nM/400nM, NH4Cl 30mM or control and imaged live. Endosomal pH is increased upon addition of 44

    acidification inhibitors (p-values < e-118 for BafA1 200nM, < e-122 for BafA1 400nM, < e-223 for 45

    NH4Cl). Images along with pH maps are shown in D (and in S4C) and quantification in E (and in S4D). 46

    Enlarged regions of pH maps indicated by white boxes are shown in F. Box plot in E represents the 47

    distribution of medians of each repeat which is denoted by red dots. Violin plot indicates all the data 48

    points from repeats. Colour bar in F corresponds to the estimated endosomal pH. Control1 is 0.2% 49

    DMSO, Control2 is 0.4% DMSO and Control3 is 0% DMSO. Number of repeats ≥ 3 for each treatment 50

    and each repeat has >80 cells. 51

    Scale bar: 20µm (A) and 40µm (D). 52

    Figure 3: BafA1, NH4Cl and Chloroquine affect Spike-pseudovirus infection 53

    A: Schematic describing the experimental protocol for SARS-CoV2 Spike-pseudovirus transduction 54

    assay. 55

    (B-I): AGS cells were pre-incubated with the inhibitors (BafA1 50nM, NH4Cl 20mM, CQ 50µM and 56