Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs Against SARS-Cov-2

pathogens

Article Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2

Friedrich Hahn 1, Sigrun Häge 1, Alexandra Herrmann 1, Christina Wangen 1, Jintawee Kicuntod 1, Doris Jungnickl 1, Julia Tillmanns 1, Regina Müller 1, Kirsten Fraedrich 1 , Klaus Überla 1 , Hella Kohlhof 2, Armin Ensser 1 and Manfred Marschall 1,*

1 Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; [email protected] (F.H.); [email protected] (S.H.); [email protected] (A.H.); [email protected] (C.W.); [email protected] (J.K.); [email protected] (D.J.); [email protected] (J.T.); [email protected] (R.M.); [email protected] (K.F.); [email protected] (K.Ü.); [email protected] (A.E.) 2 Immunic AG, 82166 Gräfelﬁng, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-9131-852-6089

Abstract: Currently, human infections with the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are accelerating the ongoing spread of the pandemic. Several innovative types of vaccines have already been developed, whereas effective options of antiviral treatments still await a scientific implementation. The development of novel anti-SARS-CoV-2 drug candidates demands skillful strategies and analysis systems. Promising results have been achieved with first Citation: Hahn, F.; Häge, S.; generation direct-acting antivirals targeting the viral polymerase RdRp or the protease 3CLpro. Herrmann, A.; Wangen, C.; Such recently approved or investigational drugs like remdesivir and GC376 represent a basis for Kicuntod, J.; Jungnickl, D.; further development and optimization. Here, we establish a multi-readout assay (MRA) system that Tillmanns, J.; Müller, R.; Fraedrich, K.; enables the antiviral assessment and mechanistic characterization of novel test compounds, drug Überla, K.; et al. Methodological repurposing and combination treatments. Our SARS-CoV-2-specific MRA combines the quantitative Development of a Multi-Readout measurement of several parameters of virus infection, such as the intracellular production of proteins Assay for the Assessment of Antiviral Drugs against SARS-CoV-2. Pathogens and genomes, enzymatic activities and virion release, as well as the use of reporter systems. In this 2021, 10, 1076. https://doi.org/ regard, the antiviral efficacy of remdesivir and GC376 has been investigated in human Caco-2 cells. 10.3390/pathogens10091076 The readouts included the use of spike- and double-strand RNA-specific monoclonal antibodies for in-cell fluorescence imaging, a newly generated recombinant SARS-CoV-2 reporter virus d6YFP, Academic Editor: Anna Honko the novel 3CLpro-based FRET CFP::YFP and the previously reported FlipGFP reporter assays, as well as viral genome-specific RT-qPCR. The data produced by our MRA confirm the high antiviral Received: 30 June 2021 potency of these two drugs in vitro. Combined, this MRA approach may be applied for broader Accepted: 23 August 2021 analyses of SARS-CoV-2-specific antivirals, including compound screenings and the characterization Published: 25 August 2021 of selected drug candidates.

Publisher’s Note: MDPI stays neutral Keywords: COVID-19 pandemic; SARS-CoV-2 infection; cultured human cells; methodological develop- with regard to jurisdictional claims in ment; multi-readout assay (MRA); antiviral drug assessment; candidate drugs; anti-coronaviral treatment published maps and institutional afﬁl- iations.

1. Introduction Human infection with the severe acute respiratory syndrome coronavirus type 2 Copyright: © 2021 by the authors. (SARS-CoV-2) caused coronavirus disease 2019 (COVID-19), which was declared as a pan- Licensee MDPI, Basel, Switzerland. demic by the World Health Organization on 11 March 2020. The COVID-19 pandemic has This article is an open access article distributed under the terms and spread rapidly since then, and today more than 210 million infections and >4.4 million ≤ conditions of the Creative Commons deaths have been conﬁrmed, with an estimated mortality risk of 2.1% [1]. Although very Attribution (CC BY) license (https:// powerful vaccines have been produced within a short period, the spectrum of approved creativecommons.org/licenses/by/ and effective antiviral drugs for the prevention and treatment of disease is still missing [2,3]. 4.0/).

Pathogens 2021, 10, 1076. https://doi.org/10.3390/pathogens10091076 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 1076 2 of 17

Remdesivir (RDV) was first approved as a direct-acting antiviral drug against COVID- 19, yet its actual clinical efficacy and benefit for patients is still a matter of controversial debate [4,5]. New types of antiviral interventions, based on the generation of improved drug candidates, will likely require several years to develop. Given the urgency of the ongoing COVID-19 pandemic however, specific analysis tools and screening systems are promptly needed. Notably in this regard, SARS-CoV-2 is characterized by its unusual complexity amongst RNA viruses, not only concerning its virion structure and genomic coding ca- pacity, but specifically the variety of regulatory proteins and viral enzymes expressed in infected cells. The nonstructural proteins, including a 3-chymotrypsin-like protease (3CLpro), papain-like protease, helicase, RNA-dependent RNA polymerase (RdRp), and several more, have been recognized as attractive targets for developing antiviral drugs as well as options for drug combination and repurposing strategies [6–9]. Initial analyses point to the promising potential of the prototype inhibitors of RdRp (RDV, favipiravir, ribavirin and others [1]) as well as 3CLpro (GC376, boceprevir, ivermectin, tipranavir and others; [1,10,11]). The further development of these anti-SARS-CoV-2 strategies and the profound in vitro characterization of the experimental hits and developmental candidates is greatly dependent on the multidimensional nature and quality of test systems. These may either represent relatively simple measurements, such as viral enzymatic in vitro assays or standard cultured-cell infection tools, to be performed on large screening scales with an advantageous ease of application. Or these may represent more complex systems, offering the ability to assess antiviral drug effects, including a determination of their mode-of-action (MoA), an in vitro characterization in physiologically appropriate cell types, drug resistance profiling or proof-of-concept (PoC) studies. In this report, we describe a multi-readout system (MRA) for the measurement of the inhibitory characteristics of anti-SARS-CoV-2 compounds in a disease-relevant human cell line. This MRA system combines the quantitative and qualitative measurements of several parameters of virus infection. With the study, we intended to emphasize the high usefulness of this system for SARS-CoV-2 research that can be applied for comparative analyses of specific viral strains and may be rapidly adapted to newly emerging variants. Putative options of a current use in the ongoing COVID-19 antiviral drug research are discussed.

2. Results 2.1. Establishment of the SARS-CoV-2 Infection System in Human Caco-2 Cells for Isolate MUC-IMB-1/2020 and SARS-CoV-2 Reporter Virus In a first step of the analysis, viral stocks were produced in Caco-2 cells and their application in quantitative antiviral assays was prepared by endpoint titration experiments. For both the clinical isolate of SARS-CoV-2, MUC-IMB-1/2020 (Figure1A), and the SARS- CoV-2 reporter virus, termed d6-YFP (Figure1B), serial dilutions of the stocks were used for the infection of Caco-2 cells. Viral titers were determined by the readout of an in-cell immunofluorescence assay (mAb-S) or a reporter-based fluorescence measurement (YFP), respectively. Moreover, the viral titer of d6-YFP determined by endpoint titration was confirmed in a plaque formation assay by directly utilizing the quantitative signals of virus-induced fluorescence of the infected cell plaques (Figure1C). PathogensPathogens2021 2021, 10, 10 1076, x FOR PEER REVIEW 3 of 318 of 17

FigureFigure 1. Titration 1. Titration experiments experiments of SARS-CoV-2of SARS‐CoV stock‐2 stock viruses. viruses. (A) ( MUC-IMB-1A) MUC‐IMB isolate‐1 isolate determined determined by immunostainingby immunostaining using mAb-Susing versus mAb‐S cell versus viability cell viability indicated indicated by SYTOX by SYTOX Blue staining. Blue staining. (B) Reporter (B) Reporter virus virus d6-YFP d6‐ determinedYFP determined by reporter by reporter signal signal quantities using YFP fluorescence versus cell viability indicated by SYTOX Blue staining. Caco‐2 cells were quantities using YFP fluorescence versus cell viability indicated by SYTOX Blue staining. Caco-2 cells were cultivated in cultivated in 96‐well plates (25,000 cells/well), infected with serial dilutions of the stock viruses as indicated, and harvested 96-wellfor evaluation plates (25,000 at cells/well),48 h post‐infection infected with(h p.i.). serial Measurements dilutions of the were stock performed viruses as in indicated, the 96‐well and format harvested as foroctuplicate evaluation at 48determinations h post-infection (values (h p.i.). are Measurementsgiven as mean ± were SD, and performed one representative in the 96-well experiment format as is octuplicateshown). Positive determinations wells were (values defined are givenas fluorometry as mean ± ‐SD,positive and onewhen representative reaching significantly experiment higher is shown).background Positive signals. wells The were x‐axes defined defined as dilutions fluorometry-positive (1:0.5 M to when1:32 reaching M or 1:16 significantly k to 1:1024 k, higher respectively) background of viral signals. inoculi. The (C x-axes) Plaque defined formation dilutions assay (1:0.5of d6‐YFP. M to Caco 1:32 M‐2 cells or 1:16 infected k to 1:1024 with k, respectively)indicated dilutions of viral of inoculi. the d6‐YFP (C) Plaquereporter formation virus were assay treated of with d6-YFP. an Avicel Caco-2 overlay cells to infected limit the with spread indicated of infection. dilutions Viral of plaques were detected 3 d p.i. by fluorescence imaging. (D) Images of the two viruses were recorded with an ImmunoSpot the d6-YFP reporter virus were treated with an Avicel overlay to limit the spread of infection. Viral plaques were detected Image Analyzer for the signals of viral spike protein expression (mAb‐S), reporter expression (YFP), cell viability 3 d(HOECHST p.i. by fluorescence 33342), and imaging. an overlay (D of) Images the respective of the twosignals viruses is given were (scale recorded bars could with not an be ImmunoSpot provided as Imagea limitation Analyzer of forthis the signalstechnical of device). viral spike M, million; protein k, expression thousand. (mAb-S), reporter expression (YFP), cell viability (HOECHST 33342), and an overlay of the respective signals is given (scale bars could not be provided as a limitation of this technical device). M, million; k, thousand.

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TheThe d6 d6-YFP‐YFP reporter virus was newly generated by two-steptwo‐step Red recombination. In brief,brief, an an anonymized anonymized patient patient sample sample of of SARS SARS-CoV-2‐CoV‐2 was was used used as as a a template template for for RNA RNA-‐ specificspecific RT-PCRRT‐PCR genome genome amplification amplification (Figure (Figure2). Four 2). resulting Four resulting amplicons amplicons were assembled were assembledwith a modified with a pBeloBAC11 modified pBeloBAC11 backbone (ref backbone [12] pBeloBAC11, ([12] pBeloBAC11, GenBank GenBank accession accession U51113, U51113,New England New England Biolabs/Addgene), Biolabs/Addgene), containing containing CMV and CMV T7 promotors, and T7 promotors, the HDV ribozymethe HDV ribozymeas well as as a bGH well polyAas a bGH signal. polyA Therein, signal. the Therein, viral open the readingviral open frame reading 6 gene frame (ORF6) 6 gene was (ORF6)replaced was by anreplaced enhanced by yellowan enhanced fluorescent yellow protein fluorescent (EYFP) expressionprotein (EYFP) module expression by two- modulestep homologous by two‐step recombination homologous [recombination13]. Virus reconstitution [13]. Virus was reconstitution achieved by was transfecting achieved bya co-culture transfecting of a two co‐culture 293T lines, of two either 293T expressing lines, either human expressing ACE2 human or the viralACE2 N or protein the viral to Nproduce protein rec-SARS-CoV-2 to produce rec‐SARS d6-YFP‐CoV (d6-YFP)‐2 d6‐YFP which (d6‐ wasYFP) further which propagatedwas further inpropagated Caco-2 cells. in CacoIn the‐2 titration cells. In the settings titration of stock settings viruses, of stock the viruses, dilutions the of dilutions infectious of inoculi, infectious to beinoculi, used to in bethe used quantifiable in the quantifiable range of Caco-2 range infection, of Caco were‐2 infection, thereby were ascertained thereby for ascertained the chosen for condi- the chosentions as conditions given by the as half-maximalgiven by the half tissue-culture‐maximal tissue infectious‐culture dose infectious (TCID50 ).dose A visualization (TCID50). A visualizationof the degree of of the infection degree at of 1 infection× TCID50 atis 1 presented× TCID50 is by presented microscopic by microscopic images (Figure images1D, (Figuresee panels 1D, a-f see for panels MUC-IMB-1 a‐f for MUC and g-r‐IMB for‐1 d6-YFP, and g‐r note for d6 the‐YFP, mAb-S note signals the mAb in c-d‐S comparedsignals in cto‐d mAb-S compared plus to YFP mAb signals‐S plus in YFP j-o, signals respectively). in j‐o, respectively). Thus, both viral Thus, strains both viral were strains found were to be foundsuitable to forbe suitable the use onfor a the broader use on scale a broader of methodological scale of methodological development development in the Caco-2 in cellthe Cacosystem.‐2 cell system.

Figure 2. Schematic depiction of pBSCoV2 d6 d6-YFP‐YFP as the genetic basis to reconstitute the novel rec rec-SARS-CoV-2‐SARS‐CoV‐2 d6-YFPd6‐YFP virus. The BACmid pBSCoV2 d6 d6-YFP‐YFP encodes a a full full-length‐length SARS SARS-CoV-2‐CoV‐2 genome, in which the the open open reading reading frame frame (ORF) (ORF) 6 was replaced by thethe enhancedenhanced yellowyellow fluorescentfluorescent proteinprotein (EYFP)(EYFP) throughthrough homologoushomologous recombination.recombination. Non-structuralNon‐structural proteins (nsp) (nsp) 1 1 to to 16 16 are are encoded encoded within within the the ORFs ORFs 1a 1aand and 1b, 1b, which which are aretranslated translated by ribosomal by ribosomal frameshifting frameshifting (blue (blue dot) duringdot) during cap‐dependent cap-dependent translation. translation. Structural Structural and accessory and accessory proteins proteins are translated are translated by their by respective their respective sub‐genomic sub-genomic RNAs and include spike (S), ORF3, envelope (E), matrix (M), ORF6 to ORF9, and the nucleoprotein (N). The genome of SARS RNAs and include spike (S), ORF3, envelope (E), matrix (M), ORF6 to ORF9, and the nucleoprotein (N). The genome CoV‐2 was assembled into a modified pBeloBAC11 backbone containing the human cytomegalovirus (CMV) immediate of SARS-CoV-2 was assembled into a modified pBeloBAC11 backbone containing the human cytomegalovirus (CMV) early gene (IE) promoter‐enhancer and T7 RNA polymerase (T7) promoter, the hepatitis delta virus ribozyme (Rz) as well asimmediate the bovine early growth gene hormone (IE) promoter-enhancer (bGH) polyadenylation and T7 RNA signal. polymerase Cm, chloramphenicol (T7) promoter, resistance; the hepatitis oriS, delta origin virus of viral ribozyme repli‐ cation;(Rz) as repE, well as replication the bovine initiation growth hormone factor of (bGH)E. coli; polyadenylationparA, ATPase; parB, signal. DNA Cm,‐binding chloramphenicol protein; parC, resistance; cis‐acting oriS, sequence origin of (parA/B/Cviral replication; required repE, for replicationsuccessful partitioning initiation factor of low of E.copy coli ;plasmids); parA, ATPase; CosN, parB, site for DNA-binding packaging into protein; lambda parC, phage cis-acting parti‐ cles;sequence loxP, (parA/B/Ctarget site for required specific for cleavage successful by Cre partitioning recombinase. of low copy plasmids); CosN, site for packaging into lambda phage particles; loxP, target site for specific cleavage by Cre recombinase. 2.2. Multiple Readouts of Measuring Antiviral Drug Activity for Clinically Relevant and Reporter2.2. Multiple‐Based Readouts SARS‐CoV of Measuring‐2 Viruses Antiviral Drug Activity for Clinically Relevant and Reporter-BasedNext, SARS SARS-CoV-2‐CoV‐2 infection Viruses assays were performed to assess the antiviral activity of two referenceNext, SARS-CoV-2 drugs in the infection Caco‐ assays2 system were and performed to establish to assessmultiple the readouts antiviral activityfor future of screeningstwo reference and drugs further in applications the Caco-2 system of antiviral and to drug establish analysis. multiple RDV readoutswas the first for futureFDA‐ approvedscreenings drug and for further SARS applications‐CoV‐2 treatment, of antiviral acting drug as a nucleoside analysis. RDV analog was prodrug. the first GC376 FDA- representedapproved drug an forinvestigational SARS-CoV-2 drug treatment, candidate, acting acting as a nucleoside as an inhibitor analog of prodrug. the viral GC376 main proteaserepresented 3CL anpro. investigationalHere, we applied drug antiviral candidate, measurements acting as for an inhibitorthese two of direct the‐ viralacting main an‐ tiviralsprotease (DAAs), 3CLpro .RDV Here, (Figure we applied 3A,B) and antiviral GC376 measurements (Figure 3C,D), for using these the two two direct-acting viral strains asantivirals a new approach (DAAs), RDVto compare (Figure clinically3A,B) and relevant GC376 (Figureand reporter3C,D),‐based using theSARS two‐CoV viral‐2 strainsviruses underas a new comparable approach conditions to compare of clinically in vitro relevant settings andof infection. reporter-based For d6 SARS-CoV-2‐YFP infections, viruses the appliedunder comparable readouts were conditions YFP reporter of in vitro fluorometry,settings of infection.the in‐cell For immunofluorescence d6-YFP infections, the assay ap- usingplied readoutsthe mAb‐ wereS antibody, YFP reporter and viral fluorometry, genome‐specific the in-cell RT immunofluorescence‐qPCR; for MUC‐IMB assay‐1/2020 using the the mAb-S antibody, and viral genome-specific RT-qPCR; for MUC-IMB-1/2020 the read-

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outs were the in-cell immunofluorescence assay (using both the mAb-S and the mAb-J2 double-strand RNA [dsRNA]-specific antibodies) and RT-qPCR. Neutral Red assay (NRA) was performed to dissect antiviral activities from the ranges of cytotoxicity (Figure3A–D, CC50 values > 100,000 nM for RDV as well as GC376). Importantly, the EC50 values for the individual readouts indicated that the inhibitory potential of both drugs was within a relatively consistent range of data, and thus confirmed the reliability of all measurements. For RDV, referring to either of the two viruses, the EC50 values of the readouts were (A) 7.4 ± 2.1, 14.6 ± 2.5, 4.8 ± 2.2 nM and (B) 24.4 ± 2.5, 22.6 ± 2.3, 16.9 ± 8.7 nM; for GC376, the EC50 values were (C) 17.7 ± 14.0, 21.9 ± 5.5, 22.0 ± 7.8 nM and (D) 39.1 ± 4.7, 450; ±, 53, 33.1 ± 8.2 nM. This indicated that, within a given range of biological variability, the readouts of this MRA were consistent and the comparative analysis underlined the strong antiviral in vitro efficacy of these drugs. Specifically, the parameters of viral replication addressed by the individual readouts conclusively showed the drug-mediated inhibition of SARS-CoV-2 replication in Caco-2 cells at several replicative stages, such as the reporter expression, block of intracellular production of viral proteins or RNA, virion release and viral genomic load. It should also be emphasized that the protocol based on the d6-YFP reporter virus greatly eases handling and improves capacities with higher sam-

Pathogens 2021, 10, x FORple PEER numbers REVIEW compared to the non-reporter parental virus. Thus, the MRA6 of 18 may facilitate a number of applications in anti-SARS-CoV-2 drug research.

ABd6‐YFP MUC‐IMB‐1 125% 125%

100% 100% viability viability 75% 75%

50% YFP 50% mAb‐S mAb‐S mAb‐J2 100%125%25%50%75%0% 100%125%25%50%75%0% replication/Cell RT‐qPCR replication/Cell RT‐qPCR 25% 25% 10000000,01 NRA 10000000,01 NRA Viral Viral 0% 0% 1 10 100 100010,000 10000100,000 100000 1 10 100 100010,000 10000100,000 100000 RDV [nM] RDV [nM]

EC50 [nM] CC50 [nM] EC50 [nM] CC50 [nM] YFP 7.4 ± 2.1 NRA >100,000 mAb‐S24.4 ± 2.5 NRA >100,000 mAb‐S 14.6 ±2.5 mAb‐J2 22.6 ±2.3 RT‐qPCR 4.8 ±2.2 RT‐qPCR 16.9 ±8.7

CDd6‐YFP MUC‐IMB‐1

125% 125%

100% 100% viability viability 75% 75%

50% YFP 50% mAb‐S mAb‐S mAb‐J2 100%125% replication/Cell 100%125%75% replication/Cell 25%50%75%0% 25% 25%50%0%RT‐qPCR 25% RT‐qPCR 10000000,01 NRA 10000000,01 NRA Viral Viral 0% 0% 1 10 100 100010,000 10000100,000 100000 1 10 100 100010,000 10000100,000 100000 GC376 [nM] GC376 [nM]

EC50 [nM] CC50 [nM] EC50 [nM] CC50 [nM] YFP 17.7 ±14.0 NRA >100,000 mAb‐S39.1 ± 4.7 NRA >100,000 mAb‐S 21.9 ±5.5 mAb‐J2 45.0 ±5.3 RT‐qPCR 22.0 ±7.8 RT‐qPCR 33.1 ±8.2 Figure 3. Comparative analysis of infection with the two SARS‐CoV‐2 viruses MUC‐IMB‐1 and d6‐YFP in Caco‐2 cells, Figure 3. Comparativeusing multiple analysis readouts of as infection indicated. Antiviral with the activity two of SARS-CoV-2 the two drugs RDV viruses (A,B) MUC-IMB-1and GC376 (C,D) and was assessed d6-YFP in in Caco-2 cells, using multipleparallel. readouts Caco as‐2 cells indicated. were cultivated Antiviral in 96‐well activity plates ofat 25,000 the two cells/well, drugs infected RDV with (A ,SARSB) and‐CoV GC376‐2 d6‐YFP ( C(A,D,C) or was assessed in MUC‐IMB‐1/2020 (B,D) at an MOI of 0.003 and harvested at 30 h p.i. Viral replication was determined by RT‐qPCR of viral parallel. Caco-2genomes cells were in the cell cultivated culture supernatants in 96-well as well plates as multiple at 25,000 combinations cells/well, of quantitative infected fluorescent with SARS-CoV-2detections using the d6-YFP (A,C) or MUC-IMB-1/2020fixed (cells,B,D including) at an MOI viral YFP of 0.003expression and and harvested antibody‐mediated at 30 h detection p.i. Viral of the replication indicated viral was antigens. determined Three inde‐ by RT-qPCR of pendent biological replicates were performed for both viruses MUC‐IMB‐1 and d6‐YFP, using the different readouts, and viral genomes inone the representative cell culture experiment supernatants is shown. as Measurements well as multiple were performed combinations in the 96‐well of quantitativeformat as quadruplicate ﬂuorescent determi detections‐ using the ﬁxed cells,nations including (values viral are given YFP as expression mean ± SD). Cell and viability antibody-mediated was determined from detection parallel cultures of the of uninfected indicated Caco viral‐2 cells antigens. Three independent biologicaldrug‐treated replicates for 48 h, and were is presented performed as mean for values both of triplicate viruses determinations MUC-IMB-1 ± SD. and d6-YFP, using the different readouts, and one representative experiment is shown. Measurements were performed in the 96-well format as quadruplicate determinations (values are given as mean ± SD). Cell viability was determined from parallel cultures of uninfected Caco-2 cells drug-treated for 48 h, and is presented as mean values of triplicate determinations ± SD.

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2.3. Use of Two Plasmid-Based Assay Systems to Screen for Novel Viral 3CLpro Protease Inhibitors under Noninfectious Conditions 2.3.1. The FRET CFP::YFP-Based 3CLpro Activity Assay (FRET Assay) In our attempts to utilize plasmid-based assay systems for analyzing the inhibitors of the 3CLpro protease, we first established a novel approach on the basis of cyan and yellow fluorescent proteins (CFP, YFP) in a CFP::YFP fusion construct. The fusion of CFP and YFP allowed for an energy transfer (FRET) from the excited CFP to the fused YFP leading to a measurable YFP emission. This so-called intrinsic FRET signal could be disrupted by the separation of the two fluorescent moieties, achieved in our construct by the activity of the 3CLpro. To this end, a transient expression plasmid for the FRET CFP::YFP fusion was generated in our laboratory, in which the two fluorescent moieties were linked through a 3CLpro-specific cleavage site, and used for cotransfection experiments together with the 3CLpro-encoding plasmid. The principle of this FRET assay was based on the idea that any addition of a 3CLpro-directed inhibitor, here represented by the reference compound GC376, blocked the cleavage of CFP::YFP and thus maintained the FRET signal (Figure4A ). Although the approach of using FRET-based reporter systems was not new and had been applied in various contexts, we adapted the FRET tool to 3CLpro-specific use in analyzing protease candidate inhibitors. The measurement of a series of increasing GC376 concentrations incubated on the reporter/3CLpro-cotransfected cells indeed confirmed the drug-mediated restoration of the FRET signal, corresponding to a 3CLpro-specific IC50 value of 69.4 ± 10.4 µM in this system. It should be emphasized that the levels pro of inhibitory IC50 values strongly correlated with the individual cell-based 3CL test systems (compare Figure5). These systems represented cellular enzyme-based tools in an overexpression situation that allowed for a quantitative assessment, but did not represent physiological target protein levels. For this reason, no simple transfer of the IC50 pro values determined in the 3CL -specific reporter systems was possible toward the EC50 values of the SARS-CoV-2 infection systems (compare Figure3). Thus, each of these reporter systems should be regarded as an individual unit of drug assessment, so that only a relative comparison between reference drugs and screening hits or new candidate compounds allowed the statement on drug efficacies. As another readout in this system, Western blot analysis was performed using aliquots of the total FRET assay lysates in order to verify the 3CLpro-specific cleavage of the fusion construct (Figure4C). FRET CFP::YFP T2A was a self-cleaving control construct that contained the T2A peptide [14] instead of the 3CLpro cleavage site (Figure4C, lane 13). The inhibition of cleavage products CFP and YFP was detected in a GC376 concentration-dependent manner (Figure4C, lanes 1–11). Thus, the FRET CFP::YFP-based 3CLpro activity assay was considered as a new option of in vitro compound testing and primary screening.

2.3.2. The FlipGFP-Based 3CLpro Activity Assay (FlipGFP Assay) Next, we used the recently developed FlipGFP-based 3CLpro activity assay [15–17]. This plasmid-encoded reporter assay was based on the expression of a conformation- specific version of the green fluorescent protein (GFP), which emitted fluorescence only after cleavage by the viral 3CLpro protease, as coexpressed from a separate plasmid. Ac- cording to the experimental optimization of the specific conditions of analysis, the FlipGFP assay allowed for an initial 3CLpro-directed antiviral drug screening in the absence of SARS-CoV-2 infection. Here, we optimized the protocol by utilizing the reference drug GC376. In particular, the ratio between plasmid concentrations for the expression of 3CLpro and FlipGFP had been serially varied in a scale of comparative settings (Figure S1), so that optimal ratios of 3CLpro/FlipGFP in the range of 0.6 + 1 to 0.2 + 1 could be experimentally defined. Using these specific conditions, the IC50 and IC90 values of GC376 were determined with 16.9 ± 7.6 µM and 99.3 ± 26.5 µM, respectively (Figure5A), in the absence of drug-induced cytotoxicity (CC50 >100 µM, selectivity index SI > 5). As a control, the mechanistically different anti-SARS-CoV-2 drug RDV was used in parallel and, as expected, did pro not show a 3CL inhibitory activity (Figure5B; SI 1, note that the IC 50 and CC50 values of Pathogens 2021, 10, x FOR PEER REVIEW 8 of 18

Pathogens 2021, 10, 1076 As a control, the mechanistically different anti‐SARS‐CoV‐2 drug RDV was used7 of in 17 parallel and, as expected, did not show a 3CLpro inhibitory activity (Figure 5B; SI 1, note that the IC50 and CC50 values of RDV lie within a very narrow range and an extrapolated ICRDV90 > lie2000 within μM). a This very approach narrow rangeconfirmed and an previous extrapolated data on IC the90 >anti 2000‐SARSµM).‐CoV This‐2 approach potential ofconﬁrmed GC376 [10,11], previous and data supported on the anti-SARS-CoV-2 the reliability of potential this reporter of GC376 assay [10 ,for11], comprehensive and supported studiesthe reliability of 3CL ofpro this‐directed reporter drugs. assay for comprehensive studies of 3CLpro-directed drugs.

Figure 4.4. EstablishmentEstablishment of thethe newnew FRETFRET CFP::YFP-basedCFP::YFP‐based 3CL3CLpropro activity assay. ( A) The principle of this assay systemsystem isis based on the Förster resonance resonance energy energy transfer transfer (FRET) (FRET) from from CFP CFP to to YFP. YFP. The The fusion fusion of of the the CFP::YFP CFP::YFP construct construct is linked is linked by pro pro bya 3CL a 3CL‐sensitivepro-sensitive cleavage cleavage site siteand andthus thus the intrinsic the intrinsic FRET FRET signal signal can be can disrupted be disrupted through through the activity the activity of 3CL of 3CL bypro sepaby‐ separatingrating the CFP the CFP from from the theYFP YFP moiety. moiety. (B) ( BThe) The 293T 293T cells cells were were used used for for transient transient transfection transfection with with the the reporter reporter pair FRET CFP::YFP and 3CLpro or the controls FRET CFP::YFP (FRET‐positive) or self‐cleaving FRET CFP::YFP‐T2A (FRET‐ CFP::YFP and 3CLpro or the controls FRET CFP::YFP (FRET-positive) or self-cleaving FRET CFP::YFP-T2A (FRET-negative). negative). At 4 h post‐tranfection (h p.t.), cells were treated with GC376 at indicated concentrations. At 19 h p.t., the FRET At 4 h post-tranfection (h p.t.), cells were treated with GC376 at indicated concentrations. At 19 h p.t., the FRET signal was signal was measured by a Victor X4 microplate reader. Two independent biological replicates were performed and one measuredrepresentative by a Victorexperiment X4 microplate is shown. reader. Measurements Two independent were performed biological in replicates the 96‐well were format performed as triplicate and one determinations, representative experimentvalues are given is shown. as mean Measurements ± SD. Inhibitory were activity performed of the in theGC376 96-well was formatassessed as and triplicate set in determinations,relation to cell viability values are (Neutral given asRed mean assay)± asSD. indicated. Inhibitory (C activity) Western of blot the GC376control wasstainings assessed with and mAb set‐GFP in relation and mAb to‐β‐ cellactin viability were (Neutralperformed Red as assay)depicted. as indicated. (C) Western blot control stainings with mAb-GFP and mAb-β-actin were performed as depicted.

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Figure 5. The FlipGFP FlipGFP-based‐based 3CLpro activityactivity assay assay [16] [16] was was applied to determine protease-directedprotease‐directed inhibitoryinhibitory drug activity in vitro. vitro. The 293T 293T cells cells were were used used for for transient transient transfection transfection with the reporter reporter pro pair FlipGFP and 3CLpro.. At At 4 4 h h p.t., p.t., cells cells were treated with GC376 at indicatedindicated concentrations.concentrations. At 19 h p.t.p.t. thethe GFPGFP signal signal was was measured measured by by a Victora Victor X4 X4 microplate microplate reader. reader. Inhibitory Inhibitory activity activity of the of two the two reference compounds GC376 (A) and RDV (B) was assessed as indicated and set in relation to reference compounds GC376 (A) and RDV (B) was assessed as indicated and set in relation to cell cell viability (Neutral Red assay). One representative experiment out of three replicates (GC376) or viability (Neutral Red assay). One representative experiment out of three replicates (GC376) or two two replicates (RDV) is shown and measurements were performed in the 96‐well format as triplicate determinations,replicates (RDV) values is shown are given and measurements as mean ± SD. were performed in the 96-well format as triplicate determinations, values are given as mean ± SD.

2.4. Combinatorial Drug Assessment Using the SARS SARS-CoV-2‐CoV‐2 d6 d6-YFP‐YFP Reporter System Additionally, thethe combinatorial combinatorial drug drug treatment treatment of SARS-CoV-2of SARS‐CoV infection‐2 infection was was analyzed ana‐ lyzedusing using the d6-YFP the d6 reporter‐YFP reporter system. system. To this To end, this the end, Loewe the Loewe additivity additivity ﬁxed-dose fixed assay‐dose was as‐ sayperformed was performed under conditions under conditions applied from applied our previousfrom our studies previous [18 studies]. Essentially, [18]. Essentially, the cotreat- thement cotreatment with RDV with plus GC376RDV plus was GC376 expected was to expected have a non-antagonistic, to have a non‐antagonistic, possibly additive possibly or additivesynergistic or effect,synergistic considering effect, considering the fact that the fact two that drugs the acted two indrugs mechanistically acted in mechanisti different‐ callymodes different of action modes (MoA), of i.e.,action a nsp12 (MoA), replication i.e., a nsp12 complex-directed replication complex MoA by‐directed nucleoside MoA ana- by nucleosidelog RDV, and analog a 3CL RDV,pro-directed and a 3CL MoApro by‐directed the protease MoA inhibitorby the protease GC376. inhibitor The results GC376. obtained The resultsfrom three obtained experimental from three replicates experimental actually replicates indicated actually that no indicated synergism that was no detectablesynergism wasupon detectable the cotreatment upon the with cotreatment RDV and GC376 with RDV (Figure and6 A–C,GC376 Replicates (Figure 6A–C, 1–3). The Replicates CompuSyn 1–3). Thealgorithm-based CompuSyn algorithm analysis of‐based the data analysis asserted of the an additivedata asserted effect an of thisadditive combination effect of treat- this combinationment (Figure 6treatmentA, mean CI (Figurewt of 1.4). 6A, However,mean CIwt part of of1.4). these However, data also part hinted of these at antagonistic data also hintedinterference at antagonistic (in particular interference CIwt of (in 1.68 particular in Replicate CIwt 2, of Figure 1.68 in6 B),Replicate as there 2, seemedFigure 6B), to be as thereno perfect seemed separation to be no perfect between separation the MoAs between of these the two MoAs drugs, of these at least twoin drugs, vitro. Theat least latter in vitro.may be The an latterin vitro mayeffect be an only in vitro arising effect under only the arising chosen under experimental the chosen conditions. experimental It maycon‐ ditions.likewise It be may based likewise on a negative be based drug on a interferencenegative drug in termsinterference of cellular in terms uptake of cellular or stability, up‐ takeor, alternatively, or stability, or, on alternatively, secondary drug on secondary targeting effects. drug targeting Nevertheless, effects. given Nevertheless, the complexity given theof coronaviral complexity gene of coronaviral regulation and gene genome regulation replication, and genome this question replication, should this be taken question into shouldcloser consideration be taken into closer before consideration addressing this before type addressing of combination this type treatment of combination in vivo. treat‐ ment in vivo.

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A mean CIwt Replicate 1 1.4 ±0.4

125% RDV CI50 1.39 GC3760%25%50%75%100%125% 100% CI75 1.47 0,000110RDV + GC376 CI 1.56 75% 90 expression) CI95 1.63 replication

50% YFP

Viral 25% CIwt 1.55 (viral 0% 110100 compound concentration [nM] B Replicate 2

125% RDV CI50 1.89 GC3760%25%50%75%100%125% 100% CI75 1.77 0,000110RDV + GC376 CI 1.67 75% 90 expression) CI95 1.60 replication

50% YFP

Viral 25% CIwt 1.68 (viral 0% 1 10 100 compound concentration [nM] C Replicate 3

125% RDV CI50 1.37 GC3760%25%50%75%100%125% 100% CI 1.13 0,000110RDV + GC376 75 75% CI90 0.94 expression) CI95 0.82 replication

50% YFP

Viral 25% CIwt 0.97 (viral 0% 1 10 100 compound concentration [nM] Figure 6. Combinatorial drug assessment of RDV and GC376 using the rec-SARS-CoV-2rec‐SARS‐CoV‐2 d6-YFPd6‐YFP reporter system. Caco Caco-2‐2 cells were cultivatedcultivated inin 96-well96‐well platesplates atat 25,00025,000 cells/well, cells/well, infectedinfected withwith SARS‐CoV‐2 d6‐YFP at an MOI of 0.003 and treated with RDV, GC376 or a combination of the drugs, SARS-CoV-2 d6-YFP at an MOI of 0.003 and treated with RDV, GC376 or a combination of the drugs, starting at the respective 4 × EC50 concentrations of the single compounds. Viral replication was starting at the respective 4 × EC concentrations of the single compounds. Viral replication was determined as 30 h p.i. by quantitative50 fluorescence detection of virus‐driven YFP expression in the determinedfixed cells. Inhibitory as 30 h p.i. profiles by quantitative of viral replication fluorescence measured detection through of virus-driven virus‐encoded YFP expressionYFP reporter in theexpression fixed cells. for InhibitoryReplicate 1 profiles (A), Replicate of viral replication2 (B) and Replicate measured 3 through (C) are virus-encoded presented as dose YFP reporterreponse expressionplots for RDV, for Replicate GC376 and 1 (A the), Replicate combination 2 (B )RDV and Replicate+ GC376. 3Three (C) are independent presented as biological dose reponse replicates plots forwere RDV, performed GC376 and as theshown, combination and measurements RDV + GC376. were Three performed independent in biological the 96‐well replicates format were as performedquadruplicate as shown,determinations, and measurements values are given were performedas mean ± SD. in the The 96-well combinatorial format asdrug quadruplicate assessment determinations,was calculated by values using are the given CompuSyn as mean algorithm.± SD. The CI combinatorial values at 50, drug 75, 90 assessment and 95% virus was calculatedinhibition bywere using used the for CompuSyn the final evaluation. algorithm. CI values at 50, 75, 90 and 95% virus inhibition were used for the final evaluation. 2.5. Analysis of the Anti‐SARS‐CoV‐2 Activity of CDK Inhibitors Determined by Multiple 2.5.Readouts Analysis of the Anti-SARS-CoV-2 Activity of CDK Inhibitors Determined by Multiple Readouts In order to investigate the potential of host‐directed antivirals (HDAs), as a second exampleIn order of the to application investigate of the this potential MRA system, of host-directed we analyzed antivirals three (HDAs),inhibitors as of a cellular second examplecyclin‐dependent of the application kinases (CDKs). of this These MRA system,CDK inhibitors, we analyzed i.e., SNS three 032, inhibitors R25/alsterpaullone of cellular

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cyclin-dependent kinases (CDKs). These CDK inhibitors, i.e., SNS 032, R25/alsterpaullone Pathogens 2021, 10, x FOR PEER REVIEW 11 of 18 and LDC4297 that were directed against CDK9, CDK1/2/5 or CDK7, respectively, had previously been characterized for their antiviral properties by using a number of different virus systems [18–23]. Despite the broad in vitro and in vivo antiviral activities reported forand LDC4297,LDC4297 that which were directed included against human CDK9, and CDK1/2/5 animal or herpesviruses, CDK7, respectively, vaccinia had pre virus,‐ human viously been characterized for their antiviral properties by using a number of different adenovirusvirus systems type[18–23]. 2, humanDespite the immunodeficiency broad in vitro and in virus vivo typeantiviral 1 and activities influenza reported A virus [19], in thefor LDC4297, present analysiswhich included LDC4297 human did and not animal inhibit herpesviruses, SARS-CoV-2 vaccinia replication virus, human at non-cytotoxic concentrationsadenovirus type 2, (Figure human7 A).immunodeficiency However, in contrastvirus type to 1 LDC4297,and influenza the A CDK9 virus [19], inhibitor in SNS 032 potentlythe present inhibited analysis SARS-CoV-2LDC4297 did not replication inhibit SARS with‐CoV EC‐502 replicationvalues of 0.155at non±‐cytotoxic0.068, 0.196 ± 0.032 andconcentrations 0.193 ± (Figure0.037 µ7A).M However, when using in contrast YFP, to mAb-S LDC4297, or the mAb-J2 CDK9 inhibitor as the respectiveSNS 032 readouts potently inhibited SARS‐CoV‐2 replication with EC50 values of 0.155 ± 0.068, 0.196 ± 0.032 (Figure7B). Cell viability measurements revealed a CC 50 value of 15.4 µM resulting in and 0.193 ± 0.037 μM when using YFP, mAb‐S or mAb‐J2 as the respective readouts (Fig‐ an SI from approximately 80 to 99. Similarly, the inhibition of CDK1/2/5 by R25 reduced ure 7B). Cell viability measurements revealed a CC50 value of 15.4 μM resulting in an SI thefrom SARS-CoV-2 approximately replication 80 to 99. Similarly, efficiency the inhibition with EC of50 CDK1/2/5values of by 0.271 R25 reduced± 0.208, the 0.438 ± 0.091 andSARS 0.449‐CoV‐±2 replication0.114 µM efficiency for the respectivewith EC50 values readouts of 0.271 (Figure ± 0.208,7 0.438C). The ± 0.091 CC and50 value0.449 for R25 was determined± 0.114 μM for asthe 74.9respective± 14.2 readoutsµM resulting (Figure 7C). in The an CC SI50 from value approximately for R25 was determined 167 to 276. Taken together,as 74.9 ± 14.2 the μ MRAM resulting consistently in an SI demonstratedfrom approximately an independence 167 to 276. Taken of together, SARS-CoV-2 the replication fromMRA consistently CDK7 activity, demonstrated whereas an the independence inhibition of of SARS CDK1/2/5‐CoV‐2 replication or CDK9 from exerted CDK7 a pronounced anti-SARS-CoV-2activity, whereas the activity inhibition in of vitro. CDK1/2/5 or CDK9 exerted a pronounced anti‐SARS‐ CoV‐2 activity in vitro.

YFP0%25%50%75%100%125%mAb‐S mAb‐J2 NRA A

125% EC50 [µM] viability 100% YFP >0.8 µM 75% mAb‐S>0.8 µM mAb‐J2 >0.8 µM 50%

25% CC50 [µM] replication/Cell

NRA 5.22 ±1.49 0% 0.0010,0010.01 0,010.1 0,1 1 10100 LDC4297 [µM]

B 125% EC50 [µM] viability 100% YFP 0.155 ±0.068 75% mAb‐S 0.196 ±0.032 mAb‐J2 0.193 ±0.037 50%

25% CC50 [µM] replication/Cell NRA 15.4 ±4.0 0%

Viral 0.0010,0010.01 0,010.1 0,1 1 10100 SNS 032 [µM]

C 125% EC50 [µM] viability Viral 100% YFP 0.271 ±0.208 75% mAb‐S 0.438 ±0.091 mAb‐J2 0.449 ±0.114 50%

25% CC50 [µM] replication/Cell

NRA 74.9 ±14.2 0%

Viral 0.0010,0010.01 0,010.1 0,1 1 10100 R25 [µM] Figure 7. 7. AntiviralAntiviral activity activity of selected of selected CDK inhibitors CDK inhibitors in SARS‐CoV in SARS-CoV-2‐2 d6‐YFP‐infected d6-YFP-infected Caco‐2 cells Caco-2 cells by using multiple readouts. Caco‐2 cells were cultivated in 96‐well plates at 25,000 cells/well, in‐ byfected using with multipleSARS‐CoV readouts.‐2 d6‐YFP at Caco-2an MOI of cells 0.003 were and treated cultivated with the in 96-wellindicated platesconcentrations at 25,000 cells/well, infectedof LDC4297 with (A), SARS-CoV-2 SNS 032 (B), or d6-YFP R25/alsterpaullone at an MOI (C of). 0.003At 30 h and p.i., treated cells were with harvested the indicated and viral concentrations ofreplication LDC4297 was ( determinedA), SNS 032 by multiple (B), or combinations R25/alsterpaullone of quantitative (C). fluorescence At 30 h p.i., detections cells were using harvested and the fixed cells, e.g., viral YFP expression and antibody‐mediated detection of spike protein (mAb‐S) viralor dsRNA replication (mAb‐J2). was Measurements determined were by performed multiple combinationsin the 96‐well format of quantitative as quadruplicate ﬂuorescence deter‐ detections usingminations, the values ﬁxed were cells, given e.g., as viral mean YFP ± SD. expression Cell viability and was antibody-mediated determined from parallel detection cultures of spike protein (mAb-S)uninfected or Caco dsRNA‐2 cells (mAb-J2). drug‐treated Measurements for 48 h (presented were as mean performed values of in triplicate the 96-well determinations format as quadruplicate determinations, values were given as mean ± SD. Cell viability was determined from parallel cultures of uninfected Caco-2 cells drug-treated for 48 h (presented as mean values of triplicate determinations ± SD). Up to three independent biological replicates were performed for these three CDK inhibitors and one representative experiment is shown. Pathogens 2021, 10, 1076 11 of 17

3. Discussion 3.1. Summarized Achievements in Developing a Multi-Readout Assay for the Assessment of Anti-SARS-CoV-2 Drugs Novel options of antiviral prevention and treatment against the worldwide problem of COVID-19 are urgently needed. So far, the development of anti-SARS-CoV-2 drug candidates has been hampered by the complexity of the SARS-CoV-2 genome and genetic variability as well as the unusually complex gene regulation. Moreover, the intensified coronavirus-specific experimentation in numerous laboratories revealed the challenges of building up reliable cell culture systems for the quantitative assessment of the in vitro replication of SARS-CoV-2 reference strains, clinical isolates and rapidly evolving mutants. In this study, we focused on the methodological establishment of an MRA system that, on the one hand, offers an ease of handling and, on the other hand, enables the antiviral characterization of novel compounds at various levels, including both DAAs and HDAs, as well as drug repurposing and combination treatments. This MRA system combined the quantitative measurement of the parameters of virus infection such as the intracellular production of proteins and genomes, enzymatic activities, virion production and release, with the generation of reporter systems and recombinant viruses. The novelty of our findings was demonstrated by several points: (i) human Caco-2 cells were infected with SARS-CoV-2 (clinical isolate or recombinant virus) and could be used for the MRA evaluation of drug sensitivity by immunofluorescence imaging with spike- and double-strand RNA-specific monoclonal antibodies, in-cell ELISA and virion release-specific RT-qPCR; (ii) the recombinant SARS-CoV-2 reporter virus d6-YFP proved to be highly reliable in antiviral measurements, and may also be used as a genetic basis for the generation of further site-specific reporter mutants; (iii) two plasmid-based reporter modules, i.e., the FlipGFP and FRET assays directed to the viral 3CLpro enzymatic activity, were comparatively applied in this study; and (iv) the first data on drug combination treatment were collected for the combined in vitro administration of inhibitors directed to the RdRp and 3CLpro. The focus on Caco-2 cells in this study underlined previous reports illustrating that the in vitro efficacy of antiviral compounds analyzed for SARS-CoV-2, at least in part, has shown a substantial variation dependent on the individual cell type, test conditions and readouts (ref [24] and references therein). Specifically, the EC50 values determined in human cells compared to non-human primate Vero cells revealed marked quantitative differences for distinct compounds, a variation that could be assigned to differences in cellular transport systems, drug uptake or metabolism [25]. In the present study, we were able to characterize the optimized conditions of the MRA system exerting a high degree of stability within the Caco-2 cell model, a basic quality that is of high importance for the continued preclinical/clinical development of antiviral drugs. All of this considered, this study strongly supports the ongoing antiviral drug analysis in the face of the COVID-19 pandemic. Despite the success of vaccinations and hygienic measures, an effective SARS-CoV-2 antiviral treatment is urgently needed. Antiviral treatment might also reduce viral shedding and mitigate the spread of infection, but is primarily needed to limit disease progression and decrease both the morbidity and mortality of SARS-CoV-2 infections. Our methodological study, which focused on the development of diverse options of MRAs, reporter modules and the use of different viral strains, may facilitate the identification of SARS-CoV-2 inhibitory drug candidates, which can hopefully then be rapidly translated into clinical use.

3.2. Future Research Directions By the use of the methodological platform described in the present report, further options for a variety of different viral strains and situations of infection may be provided with an extended series of recombinant SARS-CoV-2 reporter viruses currently generated in our laboratory (Herrmann, Ensser et al., manuscript in preparation). These additional viral reporter constructs were characterized by variable sites of different reporter insertion Pathogens 2021, 10, 1076 12 of 17

within the viral genome for individual purposes. Such SARS-CoV-2 molecular clones shall be further developed with the aim to facilitate antiviral drug analyses, studies of virus tropism and the development of further vaccine candidates. In particular, such engineered adaptations may be designed in response to newly emerging pathogenic SARS- CoV-2 variants of concern (VOCs). In addition, the expected challenges associated with the occurrence of antiviral drug resistance [26,27] as well as vaccine escape mutants [28,29] may be experimentally investigated via this methodology. In summary, on the basis of the technological achievements, future emerging issues of medical impact imposed by the SARS-CoV-2 pandemic may be addressed in a more efﬁcient and focused manner.

4. Materials and Methods 4.1. Cell Culture and SARS-CoV-2 Infection ◦ Human Caco-2 cells were cultivated at 37 C, with 5% CO2 and 80% humidity using Dulbecco’s modified Eagle medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2 mM GlutaMAXTM (35050038, Thermo Fisher Sci- entific), 10 µg/mL gentamycin (22185.03, SERVA, Heidelberg, Germany), 10% fetal bovine serum (FBS, F7524, Sigma Aldrich, St. Louis, MO, USA) and 1% MEM Non-Essential Amino Acids Solution (11140050, Thermo Fisher Scientific). SARS-CoV-2 (MUC-IMB-1/2020, passage ER-P2-2, Bundeswehr Institute of Microbiology, Munich, Germany), initially prop- agated on Vero E6 cells (ATCC® CRL-1586), was passaged twice in Caco-2 cells before being used for the infection of these cells. For generation of virus stocks, supernatants were harvested after 50 h and aliquots were stored at −80 ◦C. Viral titers were determined by endpoint titration on Caco-2 cells. For infection experiments, 25,000 Caco-2 cells were seeded in 96-well plates and inoculated with SARS-CoV-2 at an MOI of 0.003. Infection volumes of 200 µL contained antiviral test compounds or solvent control. Supernatants and cells were harvested 30 h p.i.. For further downstream analysis, cells were washed with PBS and fixed with 10% formalin at room temperature overnight. Viral supernatants were inactivated for 10 min at 95 ◦C in sealed plates. Viral replication was assessed by variable readouts as indicated and was presented as mean values of biological quadruplicates ± SD. All infection experiments were performed under BSL-3 conditions.

4.2. Cloning and Reconstitution of Rec-SARS-CoV-2 d6-YFP An anonymized residual sample from a patient with SARS-CoV-2 infection was used as a template for genome amplification. Total nucleic acids of a respiratory swab sample were extracted on an automated Qiagen EZ1 analyzer using the Qiagen EZ1 virus mini kit v2.0 according to the manufacturer’s instructions. Genomic viral RNA was reverse transcribed using the LunaScript RT SuperMix Kit (NEB) according to the manufacturer’s protocol. Four overlapping fragments covering the entire viral genome were amplified using the Q5 High-Fidelity DNA Polymerase (NEB). The resulting amplicons were assembled with a pBeloBAC11 backbone (NEB, GenBank Accession #: U51113) modified with CMV and T7 promotors and a bGH polyA signal (amplified from pcDNA4, Thermo Fisher Scientific, V86320) as well as the HDV ribozyme (synthesized as gene block by Integrated DNA Technologies IDT) using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). Assembled DNA was electroporated into the E. coli GS1783 strain and the resulting clones designated pBSCoV-2 were confirmed by restriction digestion and next-generation sequencing (MiSeq™, Illumina). The viral ORF6 gene was replaced with EYFP by homologous recombination using the two-step Lambda-Red Recombination System [13]. Positive clones of pBSCoV2 d6-YFP were confirmed by restriction digestion and next generation sequencing. For virus reconstitution, a co-culture of HEK293T cells stably expressing either ACE2 (ref [30] cloned into pLV-EF1a-IRES-Blast, Addgene #85133) or the viral N protein (amplified from patient material; cloned into pLV-EF1a-Blast) and T7-RNA polymerase (amplified from pCAGT7, kindly provided by Marco Thomas, Virology, FAU, Erlangen; cloned into pLV-EF1a-IRES-Puro, Addgene #85132) was transfected with pBSCoV2 d6-YFP using GenJet™ Reagent (II) (SignaGen®) according to the manufacturer’s protocol. At three Pathogens 2021, 10, 1076 13 of 17

d p.t., the supernatant was transferred onto Caco-2 cells for passage 1 (P1) virus stocks. Passage 2 (P2) virus stocks were obtained after infection of Caco-2 cells with 1:50 dilution of P1 virus. Viral titers of rec-SARS-CoV-2 d6-YFP were determined by endpoint titration on Caco-2 cells.

4.3. Plaque Formation Assay Caco-2 cells were seeded in 6-well plates to grow confluent monolayers and were used for the infection with serial dilutions of the d6-YFP reporter virus. At two h p.i., the inoculum was removed and replaced with medium containing 1.5% Avicel RL-591 (IFF Nutrition & Biosciences, Oegstgeest, The Netherlands). Cells were incubated for 3 days and fixed by the addition of an equal volume of 8% PFA in PBS for at least 2 h at 4 ◦C. Afterwards, the overlay was carefully removed, cells were washed at least three times with PBS to completely remove the overlay medium, before fluorescent virus plaques were imaged with an Advanced Fluorescence Imager (Intas Science Imaging, Göttingen, Germany).

4.4. Antiviral Compounds RDV (Gilead, Foster City, CA, USA) and GC376 (TargetMol, Boston, MA, USA) were used as reference compounds to assess the anti-SARS-CoV-2 in vitro activity or viral 3CLpro protease activity, respectively. The CDK inhibitors LDC4297 (Lead Discovery Center, GmbH, Dortmund, Germany), SNS 032 (Tocris, Bristol, United Kingdom) and R25 (also termed alsterpaullone, GPC Biotech AG, Martinsried, Germany) were obtained from indicated sources.

4.5. RT-qPCR for the Detection of Extracellular SARS-CoV-2 For measurements with virus-specific RT-qPCR, inactivated viral supernatants were digested with proteinase K (final concentration of 0.136 mg/mL) for 1 h at 56 ◦C followed by ◦ 5 min heat inactivation at 95 C and a dilution of 1:10 in H2O. For further analysis, volumes of 5 µL of the digested supernatants were used and the RT-qPCR was performed according to AgPath-ID™ One-Step RT-PCR (AM1005, Thermo Fisher Scientific) or NEB Luna Universal Probe One-Step RT-qPCR (E3006, NEB). Primer sequences were adapted from Corman et al., 2020 ([31], RdRp_SARSr-F and RdRp_SARSr-R). The probe (caggtggaacct- catcaggagatgc) was 50 labeled with 6-FAM (6-carboxyfluorescein) and 30 with BHQ-1 (Black Hole Quencher 1). All oligonucleotides were purchased from Biomers.net (Ulm, Germany).

4.6. In-Cell Immunostaining for the Detection of Intracellular SARS-CoV-2 In-cell immunostaining for assessment of SARS-CoV-2 replication was performed similar to previously described experimental approaches [24] by implementing fluorescence labels to facilitate the highly quantitative detection of different antigens as well as microscopic imaging of infected cells. Formalin-fixed cells were permeabilized with 0.2% Triton X-100 in PBS and blocked with blocking buffer (1% BSA in PBS). Cells were sequentially incubated with primary and secondary antibodies diluted in blocking buffer, with two PBS washing steps after each antibody incubation. A new mouse monoclonal against the viral spike (S) protein (mAb-S TRES-6.18) was produced as described previously and used for staining the viral S protein (in-cell immunofluorescence protein-specific assay) [24]. Double-stranded RNA (in-cell immunofluorescence dsRNA-specific assay) was detected by the mAb J2 (SCIONS) [32] specifically recognizing RNA helices of at least 40 bp while being inert towards other nucleic acids present in uninfected cells. Alexa 647- or Alexa 488-labeled goat anti-mouse secondary antibodies were used for quantitative detection or microscopic imaging, respectively (anti-mouse Alexa 647, A-21236, ThermoFisher Scientific; anti-mouse Alexa 488, A11029, ThermoFisher Scientific). For quantitative detection of Alexa 488 in the context of cells harboring YFP fluorescence, bound antibodies were eluted by incubating the antibody-stained cells with 62.5 mm Tris pH 6.8, 100 mm β-mecaptoethanol and 2% SDS at 37 ◦C for 2 h [33]. Fluorescence was determined to after the transfer of the eluate in a new multiwell plate by a Victor X4 multilabel reader (Perkin Pathogens 2021, 10, 1076 14 of 17

Elmer) to allow detection of Alexa 488 in the absence of YFP. Cell layers were used for subsequent restaining procedures after several washing steps with PBS. For microscopic analyses of cells displaying endogenous YFP ﬂuorescence, viral antigens were stained with an appropriate primary antibody followed by an Alexa 647-conjugated goat secondary antibody and imaged by an ImmunoSpot® S6 ULTIMATE UV Image Analyzer (Cellular Technology Limited/CTL, Cleveland, OH, USA). Parallel to antibody staining, one of the two ﬂuorescent DNA dyes SYTOX Blue or Hoechst 33342 (both ThermoFisher) was used as an internal control for estimating cell counts in Victor X4 or the ImmunoSpot reader measurements, respectively.

4.7. Two Plasmid-Based Approaches to Analyze SARS-CoV-2-Specific Protease Inhibitors: The New FRET CFP::YFP-Based 3CLpro Activity Assay (FRET Assay) and the Recently Established FlipGFP-Based 3CLpro Activity Assay (FlipGFP Assay) The FlipGFP and 3CLpro expression plasmids were kindly provided by Nicholas Heaton, Duke University Molecular Genetics and Microbiology, Durham, NC, USA. Ex- pression plasmids coding for FRET CFP::YFP and FRET CFP::YFP T2A were generated by standard polymerase chain reaction (PCR) amplification. Oligonucleotide primers used for PCR were purchased from Biomers.net (Table S1, Ulm, Germany). After cleavage with the corresponding restriction enzymes, PCR products were inserted into the Flip-GFP backbone (pLEX). Both assays were performed under very similar experimental conditions. ◦ HEK 293T cells were cultivated at 37 C, with 5% CO2 and 80% humidity using Dulbecco’s modified Eagle’s medium (DMEM, 11960044, Thermo Fisher Scientific) supplemented with 1× GlutaMAX™(35050038, Thermo Fisher Scientific), 10 µg/mL gentamicin (22185.03, SERVA) and 10% fetal bovine serum (FBS, F7524, Sigma Aldrich). Cells were seeded in a 10 cm dish at about 80% confluency and transient cotransfection of the FlipGFP or FRET CFP::YFP with 3CLpro was performed with polyethylenimine-DNA complexes (Sigma- Aldrich) as described previously [34]. At 4 h p.t., cells were trypsinized and transferred onto a 96-well plate and immediately treated with GC376 or RDV at indicated concentrations. At 19 h p.t., cells were analyzed by a Victor X4 microplate reader (PerkinElmer, Waltham, MA, USA). Subsequently, a Neutral Red assay was performed as described previously [24] to determine the cytotoxicity. Western blot analysis was performed by standard procedures as described previously [35], using mAb-GFP (11814460001, Roche) and mAb-β-Actin (A5441, Sigma Aldrich, St. Louis, MO, USA) as primary staining antibodies. For FRET measurements, three fluorescent parameters were determined in parallel by using appropriate combinations of light sources and detection filters (excitation/emission given in nm) for CFP (436/480), YFP (485/353) and FRET (436/353). In the uncleaved FRET CFP::YFP construct, the excitation of CFP leads to an energy transfer to YFP and thus to an emission of YFP signal. In the cleaved construct, however, the so-called FRET signal energy transfer is disrupted in a way that no YFP is emitted any longer through the excitation of CFP. The obtained raw fluorescence values were background corrected by using untransfected cells. Resulting FRET values were normalized for the amount of total CFP and YFP fluorescence to account for potential differences in cell densities. The FRET signal for single transfected FRET CFP::YFP served as the 100% reference (no cleavage, maximal FRET). The mock-treated cotransfection of FRET CFP::YFP with 3CLpro was set to 0% (maximal cleavage, lowest observable FRET). This step also corrects the FRET signal for donor and acceptor spectral bleed through, and thus makes the parallel measurement of CFP and YFP single transfections redundant since all FRET CFP::YFP-containing samples are expected to yield comparable amounts of total CFP and YFP fluorescence.

4.8. Drug Combination Loewe Fixed-Dose Assay Adapted to SARS-CoV-2 d6-YFP Infection Loewe additivity was assessed using an adapted protocol of the HCMV GFP-based replication assay described previously [18]. Infection of Caco-2 cells was performed as described above. Concentrations of antiviral test compounds were added as twofold serial dilutions starting at 4 × EC50 as highest concentration and comprising seven dilution steps of each single compound and well as the combination thereof. All infections were Pathogens 2021, 10, 1076 15 of 17

performed in biological quadruplicates. Viral replication was assessed by YFP quantitation in a Victor X4 microplate reader (PerkinElmer, Waltham, MA, USA) as described above. Antiviral efﬁcacy (mean of biological quadruplicates) was expressed as the percentage of mock-treated control and entered into the CompuSyn software (Version 1.0; Chou, T.C.; Martin, N. 2005, CompuSyn for drug combinations; ComboSyn, Inc., Paramus, NJ, USA). CI values at 50, 75, 90 and 95% virus inhibition were used for ﬁnal evaluation.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/pathogens10091076/s1. Table S1. Oligonucleotide primers used in this study. Figure S1: Steps of optimization of the FlipGFP assay: protease/reporter ratios. Author Contributions: Conceptualization, F.H., S.H., A.H. and M.M.; methodology, F.H., S.H., A.H., C.W., J.K., D.J., J.T., R.M., K.F., A.E. and M.M.; validation, F.H., S.H., C.W., J.K., R.M. and M.M.; formal analysis, F.H., S.H., C.W., J.T., R.M. and M.M.; investigation, F.H., S.H., A.H., J.K., H.K., A.E. and M.M.; resources, K.Ü., H.K., A.E. and M.M.; data curation, F.H., S.H., A.H., C.W., J.K., J.T., R.M. and M.M.; writing—original draft preparation, F.H., S.H., A.H. and M.M.; writing—review and editing, F.H., S.H., A.H., J.K., A.E. and M.M.; visualization, F.H. and S.H.; supervision, F.H., K.Ü., H.K., A.E. and M.M.; project administration, M.M.; funding acquisition, H.K., A.E. and M.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by BMBF SenseCoV2 01KI20172A, DFG Fokus COVID-19 EN 423/7-1, a coronavirus research grant by the Bavarian State Ministry of Science and the Arts to A.E., and Bayerische Forschungsstiftung AZ-1499-21 Immunic/Marschall. Institutional Review Board Statement: Not applicable (this study does not involve humans or animals). Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article and Supplementary Material. Acknowledgments: The authors greatly acknowledge Marco Thomas for providing HEK293T cells stably expressing the SARS-CoV-2 receptor ACE2 (Virology, FAU, Erlangen), Gerhard Dobler for supplying the MUC-IMB-1 isolate (Bundeswehr Institute of Microbiology, Munich, Germany), An- tonia Sophia Peter for the generous gift of mAb-S and the additional reagents for SARS-CoV-2 analysis (Virology, FAU, Erlangen), and Konstantin Sparrer for providing Caco-2 cells and support in performing plaque assays (Virology, Univ. Ulm, Germany). In particular, we would like to thank Nicholas Heaton and his colleagues for the very helpful methodological exchange and for providing the FlipGFP assay system (Duke University Molecular Genetics and Microbiology, Durham, NC, USA). The ELISpot/ImmunoSpot® S6 ULTIMATE UV Image Analyzer (Cellular Technology Limited/CTL, Cleveland, OH, USA) was obtained with financial support from Fonda- tion Dormeur, Vaduz. This work was published to honour the memory of Bernhard Fleckenstein († 4 May 2021), specifically acknowledging his constant efforts over many years in creating the stim- ulating scientific atmosphere at the Virological Institute Erlangen. Conflicts of Interest: The authors declare no conflict of interest.

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