key: cord-1036792-8g1ef7ns
authors: Chiem, Kevin; Vasquez, Desarey Morales; Park, Jun-Gyu; Platt, Roy Neal; Anderson, Tim; Walter, Mark R.; Kobie, James J.; Ye, Chengjin; Martinez-Sobrido, Luis
title: Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes
date: 2020-11-17
journal: bioRxiv
DOI: 10.1101/2020.11.16.386003
sha: e6eff8ab0cd0f6f7f1fbaf3e0b96a4a664d77c70
doc_id: 1036792
cord_uid: 8g1ef7ns

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab), but no vaccines, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. Importance Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

Late in 2019, a previously unknown coronavirus, severe acute respiratory syndrome 72 coronavirus 2 (SARS-CoV-2), was identified in Wuhan, China (1). Since then, SARS-73

CoV-2 has become responsible for the global pandemic of coronavirus disease 2019 74 (COVID-19) (1). As of November 2020, SARS-CoV-2 has spread worldwide and it has 75 been responsible of over 40 million confirmed cases and around 1.1 million deaths (2). Sigma) was included as a loading control. Primary antibodies bound to the membrane 238 were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies 239 against mouse or rabbit (GE Healthcare). Proteins were detected by 240 chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate 241 (Thermo Scientific) based on the manufacturer's specifications and imaged in a 242

ChemiDoc imaging system (Bio-Rad). 243

Confluent monolayers of Vero E6 cells (1.2 x 10 6 cells/well, 6-well plate format, 245 triplicates) were infected with WT or reporter-expressing rSARS-CoV-2 for 1 h at 37ºC. 246

After viral absorption, infected cells were overlaid with agar and incubated at 37ºC for 247 72 h. Afterwards, cells were submerged in 10% neutral buffered formalin at 4ºC for 16 h 248 for fixation and viral inactivation, and then the agar overlays were gently removed. To 249 observe Venus and mCherry fluorescence expression, PBS was added to each well and 250 plates were imaged under a fluorescence microscope (EVOS M5000 imaging system). 251

For immunostaining, plates were permeabilized with 0.5% Triton X-100 PBS for 10 min 12 at room temperature, blocked with 2.5% BSA PBS for 1 h at room temperature, and 253 then incubated at 37ºC for 1 h using the anti-SARS 2 NP MAb 1C7. Plaques were 254 developed for visualization using the Vectastain ABC kit and DAB HRP Substrate kit 255 (Vector laboratories), in accordance to the manufacturer's recommendations. 256

Vero E6 cells (1.2 x 10 6 cells/well, 6-well plate format, triplicates) were infected (MOI 258 of 0.01) with rSARS-CoV-2/WT or rSARS-CoV-2 expressing Venus, mCherry, or Nluc. 259

After viral adsorption for 1 h at 37ºC, cells were washed with PBS, provided with fresh 260 post-infection media, and then placed in a 37ºC incubator with 5% CO 2 atmosphere. At 261 the indicated times post-infection (12, 24, 48, 72, and 96 h) , cells were imaged for 262

Venus or mCherry expression under a fluorescence microscope (EVOS M5000 imaging 263 system). Viral titers in the tissue culture supernatants at each time point were 264 determined by titration and immunostaining, as previously described, using the anti-265 SARS-CoV NP MAb 1C7. Nluc expression in tissue culture supernatants was quantified 266

using Nano-Glo luciferase substrate (Promega) following the manufacturer's 267 recommendations. Mean values and standard deviation (SD) were determined using 268 GraphPad Prism software (version 8.2). 269

Vero E6 Non-linear regression curves and the half maximal effective concentration (EC 50 ) of 295

Remdesivir was determined using GraphPad Prism software (version 8.2). 296 

The pBeloBAC11 plasmid encoding the full-length viral genome of SARS-CoV-2 was 354 previously described (19) . To generate the reporter-expressing rSARS-CoV-2, the 7a 355 open reading frame (ORF) was substituted with Venus, mCherry, or Nluc gene in the 356 pBeloBAC11 plasmid encoding the remaining viral genome to produce pBeloBAC11-357 SARS-CoV-2-del7a/Venus, -del7a/mCherry, or -del7a/Nluc plasmids for viral rescues. 358

We then used our previously described BAC-based reverse genetics approach to 359 rescue rSARS-CoV-2-Venus, -mCherry, and -Nluc ( Figure 1A) . 360

We confirmed the rescue of rSARS-CoV-2 expressing -Venus, -mCherry, or -Nluc 361 reporter genes by RT-PCR using total RNA from mock-, rSARS-CoV-2/WT-or rSARS-362

CoV-2 reporter virus-infected cells using primers specific for the viral NP, the ORF7a 363 region, or the individual reporter genes ( Figure 1B) . As expected, primers specific for 364 SARS-CoV-2 NP amplified a band of ~1260 bp from the RNA extracted from rSARS-365

CoV-2-infected but not mock-infected cells ( Figure 1B) . Amplified bands using primers 366 17 in the ORF7a region resulted in the expected ~566 bp in cells infected with rSARS-CoV-367

Venus, -mCherry and -Nluc, respectively, based on the different size of the reporter 369 genes ( Figure 1B) . Primers specific for the reporter genes only results in the RT-PCR 370 amplification of bands from cells infected with the respective reporter-expressing 371 rSARS-CoV-2 ( Figure 1B) . These results demonstrate that substitution of the viral 372

ORF7a for Venus, mCherry, or Nluc genes results in the successful recovery of rSARS-373

CoV-2 containing these reporter genes. 374

Next, we characterize the reporter-expressing rSARS-CoV-2 by evaluating the 376 expression levels of Venus, mCherry, or Nluc in cell cultures, and compared them to 377 those of cells infected with rSARS-CoV-2/WT (Figure 2) . The rSARS-CoV-2 expressing 378

Venus and mCherry were directly visualized under a fluorescence microscope ( Figure  379 2A). Indirect immunofluorescence microscopy using a MAb against SARS-CoV NP was 380 used to detect rSARS-CoV-2/WT infection (Figure 2A) . As expected, Venus or mCherry 381 expression were only observed in Vero E6 cells infected with rSARS-CoV-2 expressing 382

Venus or mCherry, respectively, but not in cells infected with rSARS-CoV-2/WT ( Figure  383 2A). Importantly, only cells infected with rSARS-CoV-2-Venus or rSARS-CoV-2-384 mCherry were detected using green or red filters, respectively (data not shown). As 385 expected, the viral NP was detected in cells infected with rSARS-CoV-2-WT, -Venus, or 386 -mCherry (Figure 2A) . Expression of Nluc in rSARS-CoV-2-Nluc-infected cells was 387 evaluated from tissue culture supernatants at 48 h post-infection ( Figure 2B) . High 388 levels of Nluc expression were detected in culture supernatants of cells infected with 389 18 rSARS-CoV-2-Nluc but not from mock or rSARS-CoV-2/WT infected cells (Figure 2B) . 390

These results demonstrate that Vero E6 cells infected with rSARS-CoV-2-Venus, -391 mCherry, or -Nluc expresses the corresponding reporter genes and that viral infections 392 can be detected by fluorescence (rSARS-CoV-2-Venus or -mCherry) or luciferase 393 (rSARS-CoV-2-Nluc) without the need of antibodies that were required for the detection 394 of rSARS-CoV-2/WT. 395

We next evaluated reporter protein expression levels by Western blot assay using 396 cell lysates from either mock, rSARS-CoV-2-WT, or rSARS-CoV-2-Venus, -mCherry, or 397 -Nluc infected cells using MAbs against the viral NP, the reporter genes, or actin as a 398 loading control ( Figure 2C ). As expected, reporter gene expression was detected in cell 399

lysates of cells infected with the respective reporter-expressing rSARS-CoV-2 but not 400 from mock or rSARS-CoV-2-WT infected cells. Viral NP expression was detected in cell 401 lysates from all virus-infected cells, but not mock-infected cells ( Figure 2C) . 402

Next, we assessed reporter gene expression over a period of 96 h in cells that were 403 mock-infected (data not shown) or cells infected with WT or reporter-expressing rSARS-404

CoV-2 (Figure 3) . Venus and mCherry expression levels were determined using 405 fluorescence microscope (Figure 3A) , while Nluc activity in tissue culture supernatants 406 from infected cells was detected using a luminometer (Figure 3B) To assess whether deletion of 7a ORF and insertion of reporter genes compromised 415 viral fitness in cultured cells, we compared growth kinetics of reporter-expressing 416 rSARS-CoV-2 to those of rSARS-CoV-2/WT ( Figure 3C) . We found all the reporter-417 expressing rSARS-CoV-2 exhibited similar growth kinetics and peak viral titers of 418 infection to that of rSARS-CoV-2/WT (Figure 3C) , suggesting that deletion of the 7a 419 ORF and insertion of the reporter genes did not significantly affect viral fitness, at least 420 in cultured cells. These results also support previous findings with SARS-CoV where 421 deletion of the 7a ORF and insertion of reporter genes did not impact viral fitness in vitro 422 (28, 29). These results were further confirmed when we evaluate the plaque phenotype 423 of the rSARS-CoV-2 expressing fluorescent reporter genes and compared them to 424 those of rSARS-CoV-2/WT ( Figure 3D) . Similar plaque sizes were observed in Vero E6 425 cells infected with rSARS-CoV-2/WT and rSARS-CoV-2 expressing Venus or mCherry 426 ( Figure 3D) . Notably, Venus-positive or mCherry-positive plaques were only detected in 427 cells infected with rSARS-CoV-2-Venus or -mCherry, respectively, and not in rSARS-428

CoV-2/WT infected cells ( Figure 3D) . Importantly, fluorescent plaques overlapped with 429 those detected by immunostaining using the SARS-CoV NP 1C7 MAb. Similar to the 430 growth kinetics data, we found no significant differences in the plaque size of reporter-431 expressing rSARS-CoV-2 compared to rSARS-CoV-2/WT ( Figure 3D) . 432

To determine the feasibility of using our reporter-expressing rSARS-CoV-2 for the 434 identification of antivirals, we evaluated the ability of Remdesivir to inhibit SARS-CoV-2 435 20 in reporter-based microneutralization assays (Figure 4) . Remdesivir has been 436 previously described to inhibit SARS-CoV-2 infection and is the only FDA-approved 437 antiviral for the treatment of SARS-CoV-2 (3, 21, 30). The EC 50 of Remdesivir against 438 rSARS-CoV-2-Venus (Figure 4A, 1.07 µM) , -mCherry ( Figure 4B, 1.78 

The genetic stability of reporter-expressing recombinant viruses is important to 456 demonstrate their viability in in vitro and/or in vivo studies. To evaluate the ability of our 457 rSARS-CoV-2 to maintain fluorescent reporter gene expression, viruses were 458 21 consecutively passaged in Vero E6 cells and Venus and mCherry expression were 459 determined by plaque assay using fluorescent microscopy ( Figure 6A) . To that end, we 460 evaluated fluorescent expression of over 40 plaques before immunostaining with an 461 anti-SARS-CoV NP MAb 1C7. We found the Venus and mCherry fluorescent 462 expression from our rSARS-CoV-2 was genetically stable with nearly 100% of the 463 plaques analyzed under a fluorescent microscope ( Figure 6A) . We also evaluated the 464 complete genome sequences of the reporter-expressing rSARS-CoV-2 used in our 465 studies (P3) with those of additional passages (P4 and P5) using next generation 466 sequencing ( Figure 6B ). In the case of rSARS-CoV-2/Venus ( Figure 6B, (Figure 6B, bottom) . It is possible that these mutations are 478 most likely due to viral adaptation to Vero E6 cells but since different mutations were 479 found in the three reporter-expressing rSARS-CoV-2, it is also possible that these provide an efficient way to track viral infections using microscopy, luciferase proteins 503 are more readily quantifiable and therefore more amenable to HTS studies (14, 15, 50) . 504 23 For this reason, in this study we generated rSARS-CoV-2 expressing fluorescent 505 (Venus and mCherry) or luciferase (Nluc) proteins (Figure 1) . These reporter genes 506 were selected based on either their distinctive fluorescent properties (Venus and 507 mCherry) or because of their small size, stability, high bioluminescence activity, and 508

Although reporter-expressing rSARS-CoV-2 similar to those reported here have and mCherry) or luciferase activity (Nluc) using a microplate reader (Figures 2 and 3) . 526

Western blot analyses using specific antibodies against each of the reporter genes 527 24 further confirm expression from their respective rSARS-CoV-2 (Figures 2 and 3) . 528

Notably, despite deletion of the 7a ORF and insertion of a reporter gene, the three 529 reporter-expressing rSARS-CoV-2 displayed similar growth kinetics and plaque 530 phenotype than their WT counterpart (Figure 3) . As expected, viral infection was 531 visualized in real time, without the need of secondary approaches (e.g. MAbs) to detect 532 the presence of the virus in infected cells. Overall, reporter gene expression displayed 533 similar kinetics that correlated with levels of viral replication, further demonstrating the 534 feasibility of using these reporter genes as a valid surrogate of assess viral infection. 535

Therapeutic treatment of SARS-CoV-2 infections is currently limited to the use of 536

Remdesivir (3), and despite significant global efforts, there is no preventative vaccine 537 for the treatment of SARS-CoV-2 infections. Notably, there is a possibility, similar to the 538 situation with other respiratory viruses (e.g. influenza), of the emergence of drug-539 resistant SARS-CoV-2 variants that will impose a significant challenge to the currently 540 ongoing COVID-19 pandemic (56). Thus, it is imperative to not only discover new 541 antivirals and other therapeutic approaches but also prophylactics for the treatment of 542 SARS-CoV-2 infections. To that end, rapid and sensitive screening assays to identify 543 compounds with antiviral activity or to assess efficacy of vaccine candidates for the 544 therapeutic and prophylactic treatment of SARS-CoV-2 infections, respectively, are 545 urgently needed. In this study, we demonstrate that reporter-expressing rSARS-CoV-2 546 represent an excellent option for the rapid identification and characterization of both 547 antivirals (Figure 4) and NAbs (Figure 5) for the therapeutic and/or prophylactic 548 treatment of SARS-CoV-2 infections. Importantly, EC 50 (antivirals) and NT 50 (NAbs) 549 obtained with our reporter-expressing viruses were comparable to those obtained using 550 25 rSARS-CoV-2/WT or described by others in the literature (17, 21, 27) , demonstrating 551 the feasibility of using our reporter-based microneutralization assays for the rapid 552 identification of antivirals or NAbs (Figures 4 and 5, respectively) . Furthermore, our 553 results indicate that reporter-expressing Venus, mCherry, and Nluc rSARS-CoV-2 are 554 stable up to 5 passages in vitro in Vero E6 cells, including expression of the reporter 555 gene (Figure 6) . To date, we have not yet conducted studies to evaluate the feasibility 556 of using these reporter-expressing rSARS-CoV-2 in vivo. It is possible, and similar to 557 other respiratory viruses, that rSARS-CoV-2 expressing reporter genes could also be 558 used to study the biology of viral infections in validated animals of viral infection. 559

Our SARS-CoV-2 reverse genetics based on the use of BAC have allowed us to 560 rescue rSARS-CoV-2/WT (19) and rSARS-CoV-2 stably expressing reporter genes. In 561 the case of our reporter-expressing rSARS-CoV-2, we removed the 7a ORF and 562 substituted it for various reporter genes without a significant impact in viral replication. Venus-, mCherry-, or WT-rSARS-CoV-2 (after IFA) was determined using fluorescent 668 images of each well and quantified using a cell image analysis software, Cell Profiler 669 (Broad Institute). Nluc activity was quantified using the Gen5 data analysis software 670 (BioTek). The 50% effective concentration (CC 50 ) of Remdesivir was determined using 671 Graphpad Prism. Dotted line indicates 50% viral inhibition. Data were expressed as 672 mean and SD from triplicate wells. Representative images (10X magnification) are 673 included. Scale bar, 300µm. 674 

P3-P5) were assessed for Venus or mCherry expression at 72 h post-infection, before 696 immunostaining with the SARS-CoV NP MAb 1C7. The percentage of reporter-697 expressing viruses was determined from ∼40-50 viral plaques per passage

750 µm) obtained from each P3-P5 viral plaques are shown. B) Sequence 700 analysis: Reporter-expressing rSARS-CoV-2 non-reference allele frequencies from 701 virus stock (P3) and after two consecutive passages in Vero cells (P4 and P5) were 702 determined using next generation sequencing, using modified rSARS-CoV-2/WT 703 reference genomes

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