key: cord-0968025-gpr86lxe authors: Bojkova, Denisa; McGreig, Jake E.; McLaughlin, Katie-May; Masterson, Stuart G.; Widera, Marek; Krähling, Verena; Ciesek, Sandra; Wass, Mark N.; Michaelis, Martin; Cinatl, Jindrich title: SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles date: 2020-04-05 journal: bioRxiv DOI: 10.1101/2020.04.03.024257 sha: a783fa59ff213e3612c455e9536c0bfeedd24742 doc_id: 968025 cord_uid: gpr86lxe SARS-CoV-2 is a novel coronavirus currently causing a pandemic. We show that the majority of amino acid positions, which differ between SARS-CoV-2 and the closely related SARS-CoV, are differentially conserved suggesting differences in biological behaviour. In agreement, novel cell culture models revealed differences between the tropism of SARS-CoV-2 and SARS-CoV. Moreover, cellular ACE2 (SARS-CoV-2 receptor) and TMPRSS2 (enables virus entry via S protein cleavage) levels did not reliably indicate cell susceptibility to SARS-CoV-2. SARS-CoV-2 and SARS-CoV further differed in their drug sensitivity profiles. Thus, only drug testing using SARS-CoV-2 reliably identifies therapy candidates. Therapeutic concentrations of the approved protease inhibitor aprotinin displayed anti-SARS-CoV-2 activity. The efficacy of aprotinin and of remdesivir (currently under clinical investigation against SARS-CoV-2) were further enhanced by therapeutic concentrations of the proton pump inhibitor omeprazole (aprotinin 2.7-fold, remdesivir 10-fold). Hence, our study has also identified anti-SARS-CoV-2 therapy candidates that can be readily tested in patients. the protein, primarily in S and the papain-like protease (nsp3) (Supplementary Table 144 1). Based on our structural analysis, we propose that 45 DCPs are likely to result in 145 structural (or functional) differences between SARS-CoV and SARS-CoV-2 proteins. A further 222 could result in some change, with our analysis suggesting that the 147 remaining 258 DCPs seem unlikely to have a substantial effect on protein structure 148 and function. The most interesting changes were detected in the spike (S) protein, which 162 mediates coronavirus entry into host cells [Cui et al., 2019; Chen et al., 2020b] . SARS-163 CoV-2 S is 77.46% sequence identical to the SARS-CoV S and most of the remaining 164 positions are DCPs (243 residues, 1%) ( by the SARS-CoV-2 residue) ( Figure 1A ). There is greater conservation around the 176 R815 cleavage site with only two DCPs in close proximity (L792=S810, T795=S813) 177 ( Figure 1A ). 178 The SARS-CoV S receptor binding domain (residues 306-527, equivalent to 179 328-550 in SARS-CoV-2) is enriched in DCPs, containing 51 DCPs (23% of residues). 180 Eleven of the 24 SARS-CoV S residues in direct contact with ACE2 were DCPs ( Figure 181 1A, Supplementary with SARS-CoV S. N439 in SARS-CoV-2 S is not present in the interface and the 191 sidechain points away from the interface ( Figure 1D ). Further, analysis of the SARS-192 CoV-2 S-ACE2 complex highlighted important roles of the V404=K417 DCP, where 193 K417 in SARS-CoV-2 S is able to form a salt bridge with ACE2 D30 ( Figure 1C ) [Yan 194 et al. Camostat inhibited cell entry of VSV pseudotypes bearing SARS-CoV-2 S in a 294 concentration-dependent manner [Hoffmann et al., 2020] . Control experiments using 295 wild-type virus were only performed using a single high camostat concentration of 100 296 µM. Here, we directly compared the concentration-dependent effects of camostat on 297 SARS-CoV-2-and SARS-CoV-induced CPE formation in Caco2 cells ( Figure 4A ). 298 Camostat displayed nearly 14-fold increased activity against SARS-CoV-2 299 (concentration that inhibits CPE formation by 50%, IC50 1.20µM) compared to SARS-300 CoV (IC50 16.7µM) ( Figure 4A ). Nafamostat is an alternative serine protease inhibitor, 301 which is approved for pancreatitis and [Minakata et Nafamostat displayed higher activity against SARS-CoV-2 CPE formation (IC50 305 0.49µM) than camostat, but similar activity against SARS-CoV (18.9µM) ( Figure 4A ). 306 Therapeutic plasma levels for both compounds were described to reach about 0.2µM 307 [Hiraku et al., 1982; Cao et al., 2008] , which is below the antivirally active 308 concentrations Testing of additional antiviral drug candidates 341 To investigate whether there are also differences in the drug sensitivity profiles 342 of SARS-CoV-2 and SARS-CoV to other antiviral drug candidates, we tested two 343 further compounds ( Figure 4A) . Previous research had shown that 344 hydroxychloroquine and ammonium chloride interfere with SARS-CoV and SARS-345 CoV-2 replication, as lysosomotropic agents that increase the pH in lysosomes [Talbot 346 and Here, we performed an in-silico analysis of the effects of differentially 382 conserved amino acid positions (DCPs) between SARS-CoV-2 and SARS-CoV 383 proteins on virus protein structure and function in combination with a comparison of 384 wild-type SARS-CoV-2 and SARS-CoV in cell culture. Our analysis identified 1243 DCPs, which represents 89% of the amino acid 386 positions that differ between SARS-CoV-2 and SARS-CoV and nearly 13% of all 387 residues encoded by the SARS-CoV genome. 258 of these DCPs (2.6% of all 388 residues) are likely to have a structural and functional impact. The DCPs are not 389 equally distributed between the proteins. DCPs are enriched in S, 3a, p6, nsp2, 390 papain-like protease, and nsp4, but very few DCPs are present in the envelope (E) 391 protein and most of the remaining non-structural proteins encoded by ORF1ab. This 392 indicates that the individual proteins differ in their tolerance to sequence changes and/ 393 or their exposure to selection pressure exerted by the host environment. 394 This large proportion of DCPs reflects the differences in the clinical behaviour 395 of SARS-CoV-2 and SARS-CoV. The mortality associated with SARS-CoV is 396 substantially higher than that associated with SARS-CoV-2 (www.who.int) [ The serine protease inhibitors camostat, nafamostat, and aprotinin inhibited 428 both SARS-CoV-2 and SARS-CoV CPE formation. In contrast to aprotinin, camostat, 429 and nafamostat exerted limited activity against double-stranded RNA formation in 430 SARS-CoV-2-infected cells. This may indicate that camostat and nafamostat rather 431 exert cytoprotective effects that prevent cells from virus-induced lysis but less 432 pronounced antiviral activity. The mechanisms underlying the enhanced anti-SARS-433 CoV-2 activity of aprotinin remain unclear. Differences in the interference with 434 additional proteases involved in SARS-CoV-2 replication may be responsible. Notably, 435 aprotinin had been identified in the past as a protease inhibitor with pronounced 436 antiviral activity, which may interfere with viral proteases in addition to cellular ones In conclusion, our in-silico study revealed a substantial number of differentially 455 conserved amino acid positions in the SARS-CoV-2 and SARS-CoV proteins. In 456 agreement, cell culture experiments identified differences in the cell tropism and drug 457 sensitivity profiles of these two viruses. Our data also show that cellular ACE2 levels 458 do not reliably indicate cell susceptibility to SARS-CoV-2. Hence, ACE2 expression 459 studies are not sufficient to predict the SARS-CoV-2 cell tropism. Differences in the 460 drug sensitivity profiles between SARS-CoV-2 and SARS-CoV, the most closely 461 related coronavirus known to have caused disease in humans, indicate that 462 approaches to identify anti-SARS-CoV-2 drugs will require testing against this virus. 463 Finally, and probably most importantly during an ongoing pandemic, we have shown 464 that the approved drug aprotinin inhibits SARS-CoV-2 infection in clinically achievable 465 concentrations. were incubated with drug for 48h. Then, 25 µL of MTT solution (2 mg/mL (w/v) in PBS) 552 were added per well, and the plates were incubated at 37 °C for an additional 4 h. 553 After this, the cells were lysed using 200 µL of a buffer containing 20% (w/v) sodium 554 dodecylsulfate and 50% (v/v) N,N-dimethylformamide with the pH adjusted to 4.7 at 555 37 °C for 4 h. Absorbance was determined at 570 nm for each well using a 96-well 556 multiscanner. After subtracting of the background absorption, the results are 557 expressed as percentage viability relative to control cultures which received no drug. 558 Drug concentrations that inhibited cell viability by 50% (IC50) were determined using 559 CalcuSyn (Biosoft, Cambridge, UK). 560 561 qPCR 562 SARS-CoV-2 and SARS-CoV RNA from cell culture supernatant samples was 563 isolated using AVL buffer and the QIAamp Viral RNA Kit (Qiagen) according to the 564 manufacturer's instructions. 565 Absorbance-based quantification of the RNA yield was performed using the 566 Genesys 10S UV-Vis Spectrophotometer (Thermo Scientific). RNA was subjected to 567 OneStep qRT-PCR analysis using the Luna Universal One-Step RT-qPCR Kit (New 568 England Biolabs) and a CFX96 Real-Time System, C1000 Touch Thermal Cycler. 569 Primers were adapted from the WHO protocol29 targeting the open reading frame for 570 RNA-dependent RNA polymerase (RdRp): RdRP_SARSr-F2 (GTG ARA TGG TCA 571 TGT GTG GCG G) and RdRP_SARSr-R1 (CAR ATG TTA AAS ACA CTA TTA GCA 572 TA) using 0.4 µM per reaction. Standard curves were created using plasmid DNA 573 (pEX-A128-RdRP) harbouring the corresponding amplicon regions for RdRP target 574 sequence according to GenBank Accession number NC_045512. For each condition 575 three biological replicates were used. Mean and standard deviation were calculated 576 for each group. 577 578 Immunostaining for double-stranded RNA 579 Immunostaining was performed as previously described [Cinatl et al., 1995 All data are provided in the manuscript and its supplements. 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