WO2021262734A2 - Fluorescent rapid neutralization assay for viral infections - Google Patents

Abstract

The present disclosure describes, in part, a fluorescent rapid neutralization assay for viral infections. A reporter system for detecting SARS-CoV-23CLpro in a sample is provided as well as methods of using the system to detect the presence of virus, to detect the presence of the viral 3CLpro protease and to screen for agents capable of inhibiting the 3CLpro protease. In some embodiments, the viral infection comprises SARS-CoV-2.

Description

FLUORESCENT RAPID NEUTRALIZATION ASSAY FOR VIRAL

INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/042,266 filed on June 22, 2020, the contents of which are incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

N/A

SEQUENCE LISTING

A Sequence Listing accompanies this application and is submitted as an ASCII text file of the sequence listing named “155554_00608_ST25.txt” which is 52.4 KB in size and was created on June 22, 2021. The sequence listing is electronically submitted via EFS-Web with the application and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

In December 2019, a novel human coronavirus (hCoV) was identified in the Hubei Province of China (1-3). The virus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes the transmissible and pathogenic disease COVID-19 (4). COVID-19 has become a global pandemic and infected over 8 million people and caused ~800,000 deaths to date (5). Current efforts to control COVID-19 are largely focused on behavioral modifications such as social distancing and the use of masks (6). These approaches attempt to slow the spread of the virus, but meaningful control of the virus will ultimately be the result of a combination of efficacious vaccines and antiviral therapeutics (7).

Antiviral therapeutics aim to disrupt the replication cycle and reduce viral load in infected individuals. Therapeutic development efforts have led to a number of candidate antiviral compounds focused mainly on two essential viral enzymes, the RNA-dependent RNA polymerase (RdRp) and the main viral protease. Remdesivir (GS-5734), recently FDA approved as an antiviral for SARS-CoV-2, targets the polymerase to suppress hCoV replication by inducing termination of RNA polymerization (8); however, the benefits of this drug in clinical trials and early use appear limited (9). Another nucleoside analogue, P-d-N4-hydroxycytidine (NHC; EIDD-1931), also inhibits SARS-CoV-2 polymerase activity, likely via inducing lethal mutagenesis of the viral genome (10). In addition to the RdRp, the viral proteases, which are critical to liberate individual viral proteins from the polyprotein produced by initial genome translation, present another attractive drug target. For SARS-CoV-2, lopinavir/ritonavir, a protease inhibitor combination, is shown to interact with the main coronavirus protease, known as 3CLpro or Mpro (11); however, early clinical trial results with these compounds have shown no significant benefits to SARS-CoV-2 patients (12). More recently, structure-based design has enabled the rapid development of new antivirals targeting the SARS-CoV-2 protease 3CLpro (13-15). At this time, these newly designed compounds are in the early stages of testing. Thus, the discovery of additional effective SARS-CoV-2 antiviral drugs remains of high importance. The identification (and subsequent improvement) of novel drugs targeting SARS-CoV-2 will require robust and high-throughput screening approaches.

SUMMARY OF THE INVENTION

The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

As SARS-CoV-2 vaccines are tested, and people are naturally infected, it will be critical to be able to measure who has generated protective immune responses. It is thought that the best protection against COVID-19 will be mediated by viral neutralizing antibodies. The current gold standard assay to measure the ability of serum to neutralize SARS-CoV-2 is a plaque assay. This technique requires 96 hours to complete.

The present disclosure provides, in part, a fluorescent reporter assay that decreases the time to measure viral neutralization to less than 24hours, and in some embodiments in as short as 8 hours. In one embodiment, the reporter comprises, consists of, or consists essentially of a modified split GFP protein that requires proteolytic activation to become fluorescent. While this assay can be used for any virus with a polyprotein that requires cleavage by a viral protease, in some embodiments, the construct is only cleavable by one of the proteases of human coronaviruses. Cell lines that are permissive to viral infection (such as Vero-E6 cells) have been stably transduced to express high levels of this split GFP protein. In the absence of viral infection, there is no fluorescent signal. Upon infection however, the viral protease is produced, the inhibitory confirmation of the split GFP is resolved by proteolytic cleavage, and the cells become fluorescent. Thus, instead of infecting cells, applying an agar overlay, and waiting for a plaque to form, the assay according to the present disclosure allows for one to simply infect cells and look for a green signal. The signal is generated quickly as the cells are pre-loaded with the split GFP reporter. In some embodiments, the split fluorescent protein may be a fusion protein with or encoded by the same construct as an additional reporter protein that may be useful as a control to ensure introduction of the construct into the cell or to normalize expression of the protease cleavable reporter. Further, in other embodiments, the assay is fixable, so infected plates can be analyzed outside of high biocontainment where infection occurs. A fluorescent reporter system is also provided in which the fluorescent reporter is incorporated into cells to form the basis for use in the assays provided herein.

In another aspect, methods of detecting SARS-CoV-2 in a sample are provided. The methods include contacting a sample comprising cells with the reporter described herein, and detecting fluorescence in the cells. The fluorescence is indicative of SARS- CoV-2 infection.

In yet another aspect, methods of detecting SARS-CoV-2 in a sample are provided. The methods include contacting the sample with the fluorescent reporter system described herein, and detecting fluorescence in the reporter system. The fluorescence is indicative of SARS CoV-2 in the sample.

In a still further aspect, methods of screening for an agent capable of inhibiting SARS-CoV-2 3CL^pro are provided. The method may include incubating the reporter systems provided herein with the agent; and detecting the reduction in fluorescence in the system. A reduction in fluorescent demonstrates the agent's ability to block viral proteolytic cleavage by 3CL^pro. Another aspect of the present disclosure provides all that is described and illustrated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figures and Examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying example figures (also “FIG ”) relating to one or more embodiments, in which:

FIG 1. AFlipGFP protease reporter with coronavirus cleavage sites fluoresces after SARS-CoV-23CLpro expression. (A) Diagram of the FlipGFP protease reporter (16) with coronavirus cleavage sequences. FlipGFP splits GFP into b1-9 and bΐq-l l, with bΐ ΐ held in parallel to bΐq by heterodimerized coiled coils E5/K5 and a linker sequence containing a coronavirus cleavage site. The CoV main protease, 3CLpro, cuts at the cleavage site, allowing bΐ 1 to “flip” antiparallel to bΐq, enabling self-assembly of the complete GFP beta-barrel and resulting in detectable fluorescence. The pan-coronavirus 3CLpro consensus sequence, LQ, is in bold. (B) Quantification of fluorescence from 293T cells 48 h after transfection with each FlipGFP reporter or superfolder GFP (sfGFP) individually and without a protease. Statistical analysis is relative to sfGFP. (C) Quantification of fluorescence from 293T cells 48 h after transfection with each FlipGFP reporter and either the SARS-CoV-2 3CLpro or an influenza virus protein (A/PR8/1834 NP). Statistical analysis is relative to NP control. (D) Images corresponding to panel C. Green, cleaved FlipGFP; blue, nuclei. Data are shown as means ± SDs (n = 3). P values were calculated using unpaired, two-tailed Student’s t tests (*, P < 0.05; **, P < 0.001; ns, not significant). Experiments were performed twice.

FIG 2. Conservation of coronavirus 3CLpro activity enables CoV protease reporter compatibility with many coronaviruses. (A) Phylogenetic tree of five coronaviruses, SARS-CoV-2, SARS-CoV, murine hepatitis virus (MHV), avian infectious bronchitis virus (IBV), and HCoV-229E, generated based on the polyprotein ORFlab using NCBI Virus (34). These viruses span three coronavirus groups: Alphacoronavirus, Betacoronavirus, and Gammacoronavirus. 3CLpro protein sequence identities are compared to the SARS-CoV-2 3CLpro. (B) Microscopy of 293T cells 48 h after transfection with CoV reporter 3 and coronavirus 3CLpro proteins or an influenza virus protein (A/PR8/1834 NP). Green, cleaved FlipGFP; blue, nuclei. (C) In black bars is quantification of the data in panel B. In blue dots are results of qPCR of CoV 3CLpro or PR8 NP RNA levels relative to untransfected cells. Data are shown as means ± SDs (n = 3); statistical analysis is relative to the NP control. P values were calculated using unpaired, two-tailed Student’ s t tests (*, P < 0.05; **, P < 0.001). Experiments were performed twice.

FIG 3. Inhibition of the SARS-CoV-2 3CLpro by the protease inhibitor GC376 is measurable with the fluorescent CoV protease reporter. (A) Microscopy of 293T cells before or 12, 24, and 48 h after transfection with CoV reporter 3 and SARS-CoV-23CLpro. Green, cleaved FlipGFP; blue, nuclei. (B) Quantification of data in panel A. Data are shown as means ± SDs (n = 3); statistical analysis is relative to the NP control. P values were calculated using unpaired, two-tailed Student’s t tests (*, P < 0.05; **, P < 0.001). (C) Quantification of 293T cells 24 h after transfection with CoV reporter 3 and SARS-CoV-2 3CLpro, with decreasing levels of 3CLpro. Data are shown as means ± SDs (n = 3); statistical analysis is relative to a 1 : 1 ratio of reporter to protease. P values were calculated using unpaired, two-tailed Student’s t tests (*, P < 0.05; **, P < 0.001). (D) In black (line and left axis label) is quantification of 293T cells 24 h after transfection with CoV reporter 3 and SARS-CoV-2 3CLpro and treatment with the pan-coronavirus protease inhibitor GC376. Data are shown as means ± SDs with nonlinear fit curve (n = 3). In blue (dots and right axis label) is calculation of cell viability relative to vehicle-only (DMSO) samples. (E) In black (line and left axis label) are results of RT-qPCR of VeroE6 cells 24 h after infection with SARS-CoV-2 at an MOI of 0.01 and treatment with the pan-coronavirus protease inhibitor GC376. Data are shown as means ± SDs with nonlinear fit curve (n = 4). In blue (dots and right axis label) is calculation of cell viability relative to vehicle-only (DMSO) samples. Data are shown as means ± SDs (n = 3). Experiments were performed twice.

FIG. 4. Modified FlipGFP construct. Nucleotide sequence of the modified FlipGFP construct used in this study. Capital letters indicate nucleotide changes introduced to silently create a new Nhel site and eliminate an existing Notl site within FlipGFP. FIG. 5. Images of background FlipGFP fluorescence. Images of 293T cells 48 hours posttransfection with each FlipGFP reporter or sfGFP. Green = fluorescing GFP, blue = nuclei. Scale bars are lOOpm. Experiment performed twice.

FIG. 6. A schematic of an example of a SARS-CoV-2 protease reporter in accordance with one embodiment of the present disclosure.

DETAILED DESCRIPTION

The present disclosure provides in one aspect the development and validation of a reporter, specifically a fluorescent reporter optimized to detect SARS-CoV-2 3CL^pro activity. This assay is performed in human cell culture and does not require biosafety level 3 (BSL3) containment. The SARS-CoV-2 reporter is based on FlipGFP, which fluoresces only after protease-mediated activation. The inventors engineered and tested three reporter constructs with distinct cleavage target sequences for activation by the SARS-CoV-2 3CL^pro. As demonstrated in the Examples, the reporter is also activatable by other coronavirus 3CL^pro proteins across subgroups (Betacoronavirus, Alphacoronavirus, and Gammacoronavirus) and host species (human, rodent, and bird). The inventors also demonstrate this reporter to test the inhibition of SARS-CoV-2 3CL^pro with a known coronavirus 3CL^pro inhibitor, GC376 (17), and then validated the correlation between reporter inhibition and inhibition of SARS-CoV-2 replication. These experiments together demonstrate the utility of this approach for the identification of novel antiviral drugs that target the SAR-CoV-2 main protease, 3CL^pro or for use in methods for rapid detection of SAR-CoV-2. This assay, or similar assays can be developed for any virus with a polyprotein that requires cleavage by a viral protease. We also developed and tested a FlipGFP with the protease site for TEV instead of the 3CL^pro cleavage site.

Reporter

The present disclosure provides a reporter for detecting SARS-CoV-2 3CL^pro in a sample. The reporter comprises a modified split fluorescent protein comprising the proteolytic site for SARS-CoV-2 3CL^pro inserted into a fluorescent protein, wherein proteolytic cleavage of the site by 3CL^pro is required for fluorescence. This reporter is able to be used to detect the presence of 3CLpro activity (e.g., SARS-CoV-2 viral protein production) within a cell. The protease cleavage site may be modified to be specific for proteases of any of the human coronaviruses or any virus that produces a polyprotein and encodes a viral protease to cleave that polyprotein.

The modified split fluorescent protein is a fluorescent protein that has been modified such that cleavage of a proteolytic site within the protein is necessary for the ability of the protein to fluoresce. In one embodiment, the reporter is based on Flip-GFP, a GFP-based fluorogenic protease reporter that works by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. See, e.g., Zhang et al. J Am. Chem. Soc. 2019, 141, 11, 4526- 4530, the contents of which are incorporated by reference regarding the Flip-GFP. The Flip-GFP of the present invention incorporates a proteolytic site for SARS-CoV-2 3CL^pro into the Flip-GFP polypeptide in order to create an inactive GFP molecule only activated by cleavage by 3CL^pro. Suitable polypeptide sequences for the 3CL^pro cleavage site include, for example, SEQ ID NO:2, 4, 6 and 8. In a preferred embodiment, the polypeptide encoding the cleavage site is SEQ ID NO:6. Suitable polynucleotides encoding SEQ ID NO:2, 4, 6 and 8 can be determined by one skilled in the art to produce the cleavage site.

In some embodiments, the reporter comprises a polypeptide for flip-3 CL^pro-GFP selected from the group consisting of SEQ ID NO: 1, 3, 5, and 7 or a polypeptide having at least 90% sequence similarity to SEQ ID NO:l, 3, 5, and 7. In some embodiments, the polypeptide may have at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence similarity to SEQ ID NO: 1, 3, 5 or 7.

In some embodiments, the reporter described herein is encoded by a polynucleotide sequence, for example, a codon-optimized polynucleotide sequence that is optimized for expression in mammalian cells, preferably human cells. For example, the reporter of the present invention may be encoded by a codon-optimized polynucleotide comprising SEQ ID NO:9, 13, 15, or 17 or a polynucleotide having at least 90% sequence similarity to SEQ ID NO: 9, 13, 15, or 17. In some embodiments, the polynucleotide may have at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence similarity to SEQ ID NO: 9, 13, 15 or 17. In another embodiment, Flip-Cherry (red fluorescent protein) can be designed using the 3CL^pro proteolytic sites described herein and the information in Zhang et al. 2019, incorporated by reference in its entirety including FlipCherry plasmid (Addgene plasmid #124436, http://n2t.net/addgene: 124436 ; RRID:Addgene_124436). Suitable polypeptide sequences can insert the 3CL^pro-cleavage site of SEQ ID NO:2, 4, 6, or 8 into the Flip- Cherry (e.g., SEQ ID NO:29,

MOT PGA YNV 1K1 DTTSHNEDXi Ml·^' VS A T.EKEVSA T.EKEVSA T.EKEVSA T.EKEVSA / / GGSGD YTFVEQ YERAEARRSTD ASX2K V S ALKEK V S ALKEKC S ALKEK V SALK EKVSALKE, wherein Xi is a linker and X2 is a 3CL^pro cleavage site of SEQ ID NO:2, 4, 6, 8). Other split fluorescent proteins are contemplated to be able to be used in this invention.

In some embodiments, a construct is provided that comprises or consists of the polynucleotide encoding the reporter described herein and a heterologous sequence. By “construct” or "nucleic acid construct" refers herein to an artificially designed nucleic acid molecule. Nucleic acid constructs may be part of a vector that is used, for example, to transform a cell. When referring to a nucleic acid molecule alone (as opposed to a viral particle, see below), the term "vector” is used herein to refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure that can be packaged into viral particles and can be expressed in dividing and non-dividing cells either extrachromosomally or integrated into the host cell genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Within the construct, the polynucleotide may be operatively linked to a transcriptional promoter (e.g., a heterologous promoter) allowing the construct to direct the transcription of said polynucleotide in a host cell. Such vectors are referred to herein as "recombinant constructs," "expression constructs," "recombinant expression vectors" (or simply, "expression vectors" or "vectors").

Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the antibodies or fragments thereof. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g. lentiviral vectors). Vector includes expression vectors, such as viral vectors ( e.g ., replication defective retroviruses (including lentiviruses), adenoviruses and adeno- associated viruses (rAAV)), which serve equivalent functions. Lentiviral vectors may be used to make suitable lentiviral vector particles by methods known in the art to transform cells in order to express the reporter described herein.

In some embodiments, the construct comprising an internal ribosomal entry site (IRES) that allows for encoding and expression from one construct of two proteins (e.g. for example the reporter and the CL3^pro enzyme, described more below). The IRES may also be useful to allow expression of an additional reporter protein. The second reporter protein may be useful to ensure introduction of the construct into the cells or to allow for normalization of expression of the split fluorescent reporter. In some embodiments, the split fluorescent protein may be a fusion protein with or encoded by the same construct as an additional reporter protein to effect these functions.

The present invention also provides a host cell comprising the isolated nucleic acids or expression vectors described herein. In some cases, nucleic acids are transfected into a non-human host cells. The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell (e.g., a prokaryotic cell, a eukaryotic cell). A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.

In some embodiments, the constructs of the present invention may be a viral vector construct capable of making viral particles that can deliver and capable of allowing expression of the reporter within the cell. In one embodiment, the vector is an adenovirus- associated viral vector (AAV) or a lentiviral vector capable of stably expressing the reporter in cells.

By “heterologous sequence” it is meant a sequence from a different species than the transgene, and can include, for example, a heterologous promoter or heterologous transcriptional regulatory region that allows for expression of the polypeptide.

"Percentage of sequence identity" or "sequence similarity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise substitutions, or additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise substitutions, additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Protein and nucleic acid sequence identities are evaluated using the Basic Local Alignment Search Tool ("BLAST"), which is well known in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al, 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula (Karlin and Altschul, 1990), the disclosure of which is incorporated by reference in its entirety. The BLAST programs can be used with the default parameters or with modified parameters provided by the user. The term "substantial identity" of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 90% to 100%. Preferred percent identity of polynucleotides can be any integer from 90% to 100%. More preferred embodiments include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The present invention also provides a host cell comprising the isolated nucleic acids or expression vectors described herein which are capable of expressing the reporter system.

In some embodiments, the reporter may be engineered to have a membrane-anchor, i.e., an anchor that tethers the reporter to the lipid membrane of the cell. Suitable membrane-anchor and methods of anchoring polypeptides to membranes are known in the art. For example, in one embodiment, the reporter is a fusion protein comprising the polypeptide sequence for SARS-CoV-2 nsp4 (SEQ ID NO: 40) or a portion thereof that allows for the reporter to be bound to the cellular lipid membrane.

Suitable methods of tethering to the lipid membrane anchor are known in the art and include, for example, glycosylphosphatidylinositol anchors, n-myristolyation (myristic acid), S-palmitolation (i.e., attachment of palmitic acid), among others. The reporter protein, such as GFP, may be fused to a transmembrane protein such that the GFP and the protease cleavage site are inside the cell membrane.

In another embodiment, the reporter comprises a split quencher fluorescent protein pair comprising the proteolytic site for SARS-CoV-23CL^pro inserted into the modified split fluorescent protein or separating a quencher fluorescent protein pair. Suitable quencher fluorescent protein pairs are known in the art and can be modified to include the SARS- CoV-2 3CL^pro (SEQ ID NO:2, 4, 6, or 8) described herein.

Fluorescent reporter system

In another embodiment, the present invention provides a fluorescent reporter system comprising a cell comprising (a) the reporter described herein. The cell can also be referred to as a host cell. The cell is capable of stable expression of the reporter as described herein. In some embodiments, the cell is a mammalian cell. In further embodiments, the cell is a human cell. In the Examples below 293T cells were used for the protease/reporter transfection assays. Other cells capable of being used in assays are known to those of skill in the art and include but are not limited to A549-Ace2, Calu3, Huh7 and Vero cells. Notably these cells may be transfected with the reporter systems provided herein using constructs and vectors available to those of skill in the art. Alternatively, the constructs may be introduced into the cells via electroporation or via transposon mediated gene delivery. As another alternative the reporter system may be incorporated into a viral vector such as a lentiviral vector and transduced into the cells. The system further comprises a culture substrate on which to grow the cells. Suitably, the substrate may be any tissue culture substrate known by one skilled in the art and preferably that would be used for high throughput screening. For example, in some embodiments, the culture substrate may be a tissue culture dish, for example, a 6-well, 12-well, 24-well, 48- well, 96-well, 384-well tissue culture dish.

In a further embodiment, the fluorescent reporter system further comprises: (b) a SARS-CoV-23CL^pro polypeptide. In other words the reporter system comprises cells that express both the reporting and the SARS-CoV-2 3CL^pro polypeptide. These cells should express the fluorescent reporter protein (e.g. the cells should be fluorescent). This system can be used to screen for compounds and agents that can inhibit SARS-CoV-2 3CL^pro proteolytic activity, and thus, inhibit SARS-CoV-2 viral protein production and propagation.

Suitable 3CL^pro polypeptides are known by those skilled in the art and include, for example, the polypeptides of SEQ ID NO: 19, 20, 21, 22 or 23 or a polypeptide having at least 90% similarity to SEQ ID NO: 19-23, alternatively, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence similarity to SEQ ID NO: 19-23. For example, the 3CL^pro polypeptide is encoded by a polynucleotide comprising SEQ ID NO: 24-28 or a sequence having at least 90% sequence similarity to SEQ ID NO:24-28, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence similarity to SEQ ID NO:24-28.

In another embodiment, the disclosure provides a method of detecting SARS-CoV- 2 in a sample. The method can comprise (a) contacting a sample with cells comprising the reporter described herein, and (b) detecting fluorescence in the cells, wherein fluorescence is indicative of SARS-CoV-2 infection.

Methods of Detecting Virus

In another embodiment, the disclosure provides a method of detecting SARS-CoV- 2 in a sample, the method comprising: (a) contacting the sample with the fluorescent reporter system described herein, and detecting fluorescence in the reporter system, wherein fluorescence is indicative of SARS CoV-2 in the sample.

The term “contacting” means that the sample comes into close proximity with cells or reporter system described herein. The term is mean to encompass in vitro and ex vivo methods. Contacting a cells includes adding the sample or agent to a cell in culture. Other suitable methods may include introducing to the cell. An advantage of the present system is that a readout can be obtained in a short period of time. For example the sample is contacted with the cell for as little as 8 hours, for example, for 8 hours or more.

The term “detecting” comprises measuring the amount of fluorescence in the cells. Suitable methods of measuring fluorescence are known in the art and include, for example, flow cytometry, fluorescent imaging, fluorimeters, basic fluorescent plate reader, among others. In some embodiments, the fluorescent signal is quantified. Suitable methods and programs to quantify fluorescence are readily known and used by one skilled in the art.

In some embodiments, the quantified amount of fluorescence correlates with the amount of virus present in the sample.

Suitable samples include a biological sample from a subject having or suspected of having a SARS-CoV-2 infection. For example, suitable biological samples include, but are not limited to, a nasal swab sample, a throat swab sample, a saliva sample, or a blood sample. Other bodily fluids are contemplated to be able to be used in the methods described herein.

Screening Method

In a further embodiment, the fluorescent reporter system described herein (e.g., cells comprising the reporter and SARS-CoV-2 3CL^pro) can be used for a high-throughput screen to identify agents capable of inhibiting SARS-CoV-2 3CL^pro. This method can also be used to test already known inhibitors of SARS-CoV-2 to see if their mechanism of action is through inhibition of 3CL^pro cleavage. The method comprises (a) incubating the reporter system that comprises both the reporter and SARS-CoV-2 3CL^pro with an agent; and (b) detecting the reduction in fluorescence in the system, wherein a reduction in fluorescent demonstrates the agent’s ability to block viral proteolytic cleavage by 3CL^pro. Suitable incubation times can be determined by one skilled in the art, for example, the agent is incubated in contact with the cell for at least 4 hours, preferably at least 8 hours or more, and then fluorescent signal is detected and measured.

In some embodiments, inhibitory curves can be determined to provide an IC50 for the agent. This can be done by (a) incubating the reporter system with different amounts of the agent; and (b) quantifying the fluorescence associated with the different amounts, wherein the fluorescence correlates with the IC50 for the agent.

Suitable agents for use in the present methods can be any biological molecule that may have viral inhibitory effects. Suitable agents include, by are not limited to, small molecules, proteins, peptides, siRNA, macromolecules, antibodies and fragments thereof, dibodies, fusion proteins, and the like. These are exemplary and the methods are not limited to any specific agents.

The present disclosure provides, in part, a fluorescent reporter assay that decreases the time to measure viral neutralization to less than 24hours, and in some embodiments in as short as 8 hours. The term “viral neutralization” refers to the ability of an agent to block the propagation of the virus in host cells.

In another embodiment, the reporter comprises, consists of, or consists essentially of a modified split fluorescent protein, such as split GFP, that requires proteolytic activation to become fluorescent. In some embodiments, the construct is only cleavable by one of the proteases of human coronaviruses. Cell lines that are permissive to viral infection (such as Vero-E6 cells) have been stably transduced to express high levels of this split GFP protein. In the absence of viral infection, there is no fluorescent signal. Upon infection however, the viral protease is produced, the inhibitory confirmation of the split GFP is resolved by proteolytic cleavage, and the cells become fluorescent. Thus, instead of infecting cells, applying an agar overlay, and waiting for a plaque to form, the assay according to the present disclosure allows for one to simply infect cells and look for a green signal. The signal is generated quickly as the cells are pre-loaded with the split GFP reporter. Further, in other embodiments, the assay is fixable, so infected plates can be analyzed outside of high biocontainment where infection occurs.

For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.

The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

As used herein, the transitional phrase "consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to "comprising."

Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g ., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The reporters, systems and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non- claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

Another aspect of the present disclosure provides all that is described and illustrated herein. The following Examples are provided by way of illustration and not by way of limitation.

Examples

Example 1: Novel reporter and high-throughput assay

In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited.

This Example provides a new reporter and assay for use in both detecting virus and for an assay for identification of new and efficacious antivirals is. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro. 3CLpro is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this Example, we demonstrate a new assay to identify inhibitors of 3CLpro. Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CLpro. This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. RESULTS

Generation of a fluorescent SARS-CoV-2 3CL^pro activity reporter.

In order to develop a fluorescent reporter responsive to the SARS-CoV-2 main protease, we started with the FlipGFP protein (16). FlipGFP is used to detect protease activity by expressing the green fluorescent protein (GFP) 10th and 11th beta-strands, bΐq- 11, separately from, and in a conformation incompatible with, the rest of the GFP beta- barrel, b 1-9 (Fig._...! A, top). A linker containing a cleavage site holds the two GFP beta- strands, bΐq-l l, in an inactive, parallel conformation. When the appropriate protease is present, the linker containing the cleavage site is cut. This cleavage allows GFP b 11 to reorient such that GFP bΐq-l l are antiparallel and able to fit into GFP b1-9, inducing fluorescence ~ 100-fold over background (16).

SARS-CoV-2 generates two proteases that cleave the viral polyprotein, a papain like protease (PL^pro) and a chymotrypsin-like protease (3CL^pro). 3CL^pro, also known as the main protease or M^pra, is the more conserved viral protease, with only 5 amino acid changes between SARS/SARS-like CoVs and SARS-CoV-2, compared to the 102 differences found in PL^pro (18). 3CL^pro cleaves at a consensus sequence, LQ|, which is highly conserved across the coronavirus family (1_.9). Additionally, 3CLpro has been shown to effectively cleave luciferase-based protease biosensors (20, 21) and fluorescence resonance energy transfer (FRET)-based probes (1_.3, 17, 22-28). With the aim of generating a protease reporter compatible with SARS-CoV-2 and other present and future coronaviruses to support viral inhibitor screening, we selected CoV 3CL^pro as our protease target.

Although CoV 3CL^pro requires the minimal consensus sequence LQ| for cleavage, the cleavage site context influences cleavage efficiency (29). Among the CoV polyprotein cleavage sites, 3CL^pro targeted sites surrounding the 3CL^pro sequence itself are generally cleaved most efficiently (3_.0). Additionally, different CoVs have distinctive optimal cleavage site sequences (24). In order to develop an efficiently cleaved CoV 3CL^pro reporter, we tested three different cleavage sequences predicted to be highly compatible with the SARS-CoV-2 3CL^pro (Fig. 1A. bottom). CoV reporter 1 contains the conserved nsp4-5 cleavage site present in the SARS-CoV and SARS-CoV-2 polyproteins (31). CoV reporter 2 contains an optimized cleavage sequence for the SARS-CoV 3CL^pro (32). CoV reporter 3 contains an optimized sequence shown to be highly cleaved by many CoV family members (24). As a negative control, we included a construct harboring the tobacco etch virus (TEV) protease cleavage site, as in the initial FlipGFP construct (16). To observe whether these FlipGFP constructs background fluoresced without CoV 3CL^pro activity, we transfected cells with each reporter or a superfolder GFP (sfGFP) expression plasmid. Compared to sfGFP, which is properly folded and fluorescing, the inactive FlipGFP constructs produced significantly lower fluorescence, from a 10- to 100-fold reduction depending on the reporter (Fig. IB; see also Fig. 5 in the supplemental material).

Our goal was to identify a construct that showed minimal background fluorescence while still being efficiently cleaved by SARS-CoV-2 3CL^pro, allowing strong fluorescence for detection via microscopy, plate reader, or flow cytometry. To test our 3CL^pro reporters, we cotransfected each reporter with a SARS-CoV-2 3CL^pro expression plasmid. At 48 h posttransfection, we could detect GFP-positive cells with each of the three CoV reporters transfected with the SARS-CoV-2 3CL^pro (Fig. 1C and D). In contrast, with transfection of a negative control, nucleoprotein from an H1N1 influenza virus (A/PR8/1934 NP), we did not detect any signal above background levels of fluorescence. Further, the reporter containing the TEV cleavage site was not activated by SARS-CoV-2 3CL^pro. Fluorescent signal across the treatment conditions demonstrated that while all three CoV reporters showed significant induction of GFP signal when coexpressed with the SARS-CoV-2 3CL^pro, CoV reporter 2 had substantial background and the level of induction with CoV reporter 1 reached only half that of the other two CoV reporters. With a 100-fold change in fluorescence and minimal background, we selected CoV reporter 3 for further testing (Fig. 1C and D).

Many CoV 3CL^pro proteins activate the FlipGFP CoV 3CL^pro reporter.

CoV 3CL^pro proteins are reasonably conserved across coronavirus groups (Fig 2 A) (33). Further, CoV reporter 3 was based on an optimized cleavage sequence for CoV 3CL^pros from each coronavirus group (24). To test whether this protease reporter was compatible with a variety of CoV 3CL^pro proteins, we expressed CoV reporter 3 with four other coronavirus proteases from different groups Alphacoronavirus , Betacoronavirus, and Gammacoronavirus) and host species (human, mouse, and bird). At 48 h after transfection, all CoV 3CL^pros induced visible fluorescence compared to the control influenza virus nucleoprotein with CoV reporter 3 (Fig 2B). Expression of the CoV 3CL^pro or PR8 NP constructs was quantified using quantitative PCR (qPCR) (Fig 20. Quantification with a plate reader demonstrated that SARS-CoV ( Betacoronavirus , human) and avian infectious bronchitis virus (IBV; Gammacoronavirus , avian) resulted in levels of fluorescence similar to that with SARS-CoV-2 ( Betacoronavirus , human) (Fig. 2(3). Murine hepatitis virus (MHV; Betacoronavirus , murine) and human coronavirus

229E (HCoV-229E; Alphacoronavirus , human) were less compatible with CoV reporter 3, while still producing 12- and 80-fold changes in fluorescence, respectively, over background (Fig 20. These experiments show that our FlipGFP 3CL^pro reporter is generally compatible with many CoV 3CL^pro proteins across coronavirus groups and host species, potentially enabling protease inhibitor screening for a variety of CoVs in addition to SARS-CoV-2.

Development of a FlipGFP CoV 3CL^pro reporter-based assay for protease inhibitor screenins in human cells. To develop an assay for protease inhibitor screening using our CoV reporter 3, we first needed to optimize the experimental conditions. We performed a transfection time course with SARS-CoV-2 3CL^pro to determine an early, appropriate time point for sample collection (Fig. 3 AT At 12 h posttransfection, only a few GFP fluorescing cells were visible and fluorescent signal was just above background (Fig. 3B). At 24 h posttransfection, green cells were visible without appreciable background signaling (Fig. 3B). At 48 h postinfection, most cells produced a high GFP signal, with some background fluorescence detectable (Fig. 3B). We therefore selected the 24-h posttransfection time point. To increase the sensitivity of our assay, we titrated the level of SARS-CoV-2 3CL^pro transfected with CoV reporter 3; our goal was to maximize activation of the reporter while minimizing the amount of protease available in the cell. We transfected cells with five ratios of reporter to protease: 1:1, 1 :0.8, 1 :0.4, 1 :0.2, and 1 :0. At 24 h posttransfection, we observed significant decreases in reporter activation at reporter-to-protease ratios of 1:0.4 and 1:0.2 (Fig 30. However, a 1:0.8 reporter-to-protease ratio resulted in no significant loss of fluorescence compared to that with a 1 : 1 ratio (Fig 3C). Based on these experiments together, we selected a 1:0.8 reporter-to-protease ratio for transfection and a 24-h posttransfection endpoint as the optimal conditions for our protease inhibitor assay using FlipGFP 3CL^pro reporter 3.

Finally, we wanted to verify that our assay could detect drug inhibition of the SARS-CoV-2 3CL^pro with a known inhibitor. Therefore, we selected a recognized pan- coronavirus 3CL^pro inhibitor, GC376, to test our assay (17). Four concentrations of GC376, that did not significantly impact cell viability compared to vehicle alone, were applied to cells at the time of transfection with CoV reporter 3 and SARS-CoV-23CL^pro. As expected, reporter activity levels were maintained at the lower protease inhibitor concentrations, while fluorescence was reduced at the higher concentrations of GC376 (Fig 3D). Thus, our assay successfully detected inhibition of SARS-CoV-3 3CL^pro by the protease inhibitor GC376. However, it is also important to verify that inhibition of our reporter is strongly correlated with inhibition of SARS-CoV-2. We infected VeroE6 cells with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 before applying protease inhibitor at the same four concentrations as tested with the protease reporter. At 24 h postinfection, we collected RNA and performed reverse transcription (RT)-qPCR to detect SARS-CoV-2 RNA; similar to the case with the reporter, viral RNA levels were suppressed in a dose-dependent manner (Fig. 3E). Our observed inhibition of the virus is consistent with reports of inhibition of SARS-CoV-2 by GC376 in the literature (22, 23). Together, these experiments demonstrate feasibility of using our FlipGFP CoV 3CL^pro reporter assay to identify protease-targeting inhibitors of SARS-CoV-2.

DISCUSSION

This Example demonstrates the development of a cell-based assay to screen for novel SARS-CoV-2 antiviral drugs at BSL2; to our knowledge, no such assay optimized for SARS-CoV-2 currently exists. Therefore, we generated a reporter requiring a coronavirus protease, 3CL^pro, for activation of a GFP fluorescent signal. We showed that this reporter is responsive to the SARS-CoV-2 3CL^pro, in addition to many different coronavirus 3CL^pro proteins. After optimizing screening conditions, we demonstrated that our reporter was sensitive to treatment with a known coronavirus protease inhibitor, GC376. These experiments illustrate the utility of our approach to identify, and subsequently optimize, novel protease inhibitors of SARS-CoV-2.

To meet the demands of virus research during the SARS-CoV-2 pandemic, reporter assays need to be flexible and high-throughput. Our reporter is activated with expression of a single CoV protein, 3CL^pro, allowing for SARS-CoV-2 drug testing at BSL2. Additionally, the reporter is compatible with many CoV 3CL^pro proteins, supporting rapid testing of inhibitors against a variety of coronaviruses, present or future, and without synthesis of protease substrates or purification of viral proteins (1_.3, 1_.7, 22-28). Further, as our assay is performed in living cells, our system enables the discovery of protease inhibitors while simultaneously evaluating effects on cellular viability. Our assay is scalable, and the analysis requires only a basic fluorescent plate reader, supporting high- throughput screening. In addition to applications in drug discovery pipelines, this assay could be deployed to determine targets of antivirals identified via viral screening. Reporter assays, including ours, also have limitations. Our reporter utilizes CoV 3CL^pro expressed alone; during a CoV infection, the protease is only one of many viral proteins present, and any inhibitors that may affect cross-viral protein interactions would be missed. Additionally, CoV infection induces significant cellular membrane rearrangements that transfection of the protease alone does not. Thus, the subcellular access of therapeutic compounds to the viral protease may fail to be reflected in our assay, and the effects of an identified protease inhibitor could significantly differ when applied to authentic viral infection. Finally, although this plasmid-based expression presents many advantages, it also necessitates further screen hit testing in the context of coronavirus infection. Although the correlation between our reporter and inhibition of viral infection was appreciable with the drug GC376, testing of more inhibitors is required to make generalizable correlations between our reporter assay and viral infection readouts.

To have the greatest impact on the COVID-19 pandemic, an effective SARS-CoV- 2 antiviral needs to be identified as early as possible. Countries around the world have taken drastic and necessary steps to limit the spread of virus, yet infection rates continue to rise in some. An antiviral treatment is unlikely to stop the spread of infection, but it is likely to limit the mortality associated with SARS-CoV-2 infection. It is our hope that this reporter assay facilitates the identification of SARS-CoV-2 protease inhibitor candidates to be rapidly optimized and translated to clinical use.

MATERIALS AND METHODS

Cell culture.

All cells were obtained from the ATCC and grown at 37°C in 5% CO2. 293T cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), GlutaMAX, and penicillin-streptomycin. VeroE6 cells were grown in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, pyruvate, nonessential amino acids (NEAA), and penicillin-streptomycin. For transfection, plates were polylysine treated and seeded with 293 Ts. Twenty-four hours later, plasmid DNA, Opti-MEM, and TransIT-LT (Mirus) were combined using pipetting and incubated at room temperature for 20 min before being added to cells by droplet. Plasmids.

Constructs (excluding CoV reporters 1, 2, and 3) were cloned into the pLEX expression vector using the BamHI and Notl restriction sites and DNA assembly (New England BioLabs [NEB]). Coronavirus 3CL^pro expression plasmids (SARS-CoV-2, SARS- CoV, MHV, IBV, and HCoV-229E) were generated using codon-optimized gBlocks (IDT). The TEV control FlipGFP (Addgene; number 124429) was designed to include a silent Nhel restriction site ahead of the TEV cleavage sequence and generated using a gBlock (IDT) (Fig. 4) with primers FlipGFP For and FlipGFP Rev (Table 1) for insertion into BamHI- and Notl-digested pLEX. The coronavirus 3CL^pro FlipGFP reporters (reporters 1, 2, and 3) were generated using forward primers containing the cleavage sequences (CoV rep 1 For, CoV rep 2 For, and CoV rep 3 For) along with the FlipGFP Rev primer (Table 1) and assembled into Nhel- and Notl-digested TEV control FlipGFP plasmid (Table 1). DNA was transformed into NEB 5-alpha high-efficiency competent cells (NEB). Insert size was verified with PCR, and purified plasmids were sequenced using Sanger sequencing.

Table 1. Primers for CoV 3CLpro responsive FlipGFP constructs

Imaging and quantification. Cells were fixed with 2% paraformaldehyde (PFA) at room temperature for 20 min before incubation in 1:10,000 Hoechst 33342 (Life Technologies) in phosphate-buffered saline (PBS) at 4°C overnight. Images were obtained using the ZOE fluorescent cell imager (Bio-Rad). Quantification was performed using the Celllnsight CX5 platform (Thermo Scientific).

CoV 3CL^pro and PR8 NP aPCR.

At 48 h after transfection of 293 Ts, RNA was prepped according to the RNeasy 96 kit (Qiagen). One-step RT-PCR was performed using primers targeting the respective CoV 3CL^pro (SARS-CoV-2, SARS-CoV, MHV, IBV, or 229E) or PR8 NP and a housekeeping gene (18S) with the iTaq universal SYBR green one-step kit (Bio-Rad) on an Applied Biosystems QuantStudio3 instrument.

Cytotoxicity assays.

At 24 h before treatment, plates were polylysine treated and seeded with 293 T or VeroE6 cells. The next day, medium was replaced with complete medium containing GC376 (MedKoo) at the desired concentrations using a constant level of a vehicle

(dimethyl sulfoxide [DMSO]). After 24 h of treatment, cells were collected according the CellTiter-GLO (Promega) protocol and luminescence levels assessed using a luminometer. SARS-CoV-2 infections and viral aPCR.

At 24 h before infection, plates were polylysine treated and seeded with VeroE6 cells. The next day, the cells were washed with PBS before infection with SARS-CoV-2 isolate USA-WA1/2020 from BEI Resources in 2% FBS-MEM infection medium at an MOI of 0.01 for 1 h. Virus was removed and cells were then placed in infection media containing GC376 (MedKoo) at the desired concentrations using a constant level of a vehicle (DMSO). At 24 h postinfection, cells were collected in TRIzol (Invitrogen), followed by RNA isolation. One-step RT-qPCR was performed with primers targeting the SARS-CoV-2 N region (BEI) using the EXPRESS one-step Superscript qRT-PCR kit (Thermo Fisher) on an Applied Biosystems QuantStudio3 instrument. RNA was normalized using an endogenous 18S rRNA primer/probe set (Applied Biosystems). REFERENCES

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34. Brister JR, Ako-Adjei D, Bao Y, Blinkova O. 2015. NCBI viral genomes resource. Nucleic Acids Res 43:D571-D577.

Example 2:

SARS-CoV-2 encodes a main viral protease, 3CL^pro, that is used to cleave the viral polyprotein, an essential task in the viral life cycle. Coronaviruses also develop double membrane vesicles as a part of their replication cycle, protecting many viral components from coming in contact with cellular proteins. In a second embodiment, a SARS-CoV-2 protease reporter tethered to the lipid membrane can be used. Suitable methods of tethering the reporter described herein to the lipid membrane anchor are known in the art and include, for example, glycosylphosphatidylinositol anchors, n-myristolyation (myristic acid), S- palmitolatio (attachement of palmitic acid), among others. For example, the reporter described herein can be made membrane-bound by fusing to the SARS-CoV-2 protein, called nsp4 which is membrane anchored. Suitable sequence of nsp4 comprises SEQ ID NO:39 for the polynucleotide sequence and SEQ ID NO: 40 for the polypeptide sequence. Insertion of a 3CL^pro-cleavage sequence at the appropriate cut site will induce fluorescence with SARS-CoV-2 infection. This technology can be used to decrease the work required to perform, as well as increase the speed and throughput of, viral neutralization assays.

In one embodiment, as depicted in Fig. 5, the reporter is a quencher and a fluorophore that will be made by separating the parts by the 3CL^pro cleavage site (e.g., SEQ ID NO: 2, 4, 6, or 8), as part of a fusion protein with a membrane bound peptide (e.g., nsp4 of SARS-CoV-2). One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

Claims

1. A reporter system for detecting SARS-CoV-2 3CL^pro in a sample, the reporter system comprising: a modified split fluorescent protein or a split quencher fluorescent protein pair comprising the proteolytic site for SARS-CoV-2 3CL^pro inserted into the modified split fluorescent protein or separating the quencher fluorescent protein pair, wherein proteolytic cleavage of the site by 3CL^pro is required for fluorescence.

2. The reporter of claim 1, wherein the reporter protein comprises:

(a) the modified split fluorescent protein is Flip GFP or FLIP-cherry; and

(b) the proteolytic site for SARS-CoV-2 3CL^pro is selected from the group consisting of: SEQ ID NO:2, 4, 6 and 8;

3. The reporter of any of the preceding claims, wherein the modified split fluorescent protein is Flip-GFP and comprises a polypeptide selected from the group consisting of SEQ ID NO:l, 3, 5, and 7 or a polypeptide having at least 90% sequence similarity to SEQ ID NO:l, 3, 5, and 7.

4. The reporter of any one of the preceding claims, wherein the reporter is encoded by a codon-optimized polynucleotide comprising SEQ ID NO: 9, 13, 15, or 17 or a polynucleotide having at least 90% sequence similarity to SEQ ID NO: 9, 13, 15, or 17.

5. The reporter of claim 4, wherein the polynucleotide is within a heterologous construct.

6. The reporter of claim 5, wherein the construct is a plasmid or a viral vector.

7. A fluorescent reporter system comprising a cell comprising:

(a) the reporter system of any one of claims 1-6.

8 The fluorescent reporter system of claim 7, wherein the cell is a mammalian cell.

9. The fluorescent reporter system of any one of claims 7-8, wherein the cell is a human cell

10. The fluorescent reporter system of any one of claims 7-9, wherein the system further comprises a culture substrate on which to grow the cell.

11. The fluorescent reporter system of claim 10, wherein the culture substrate is a 6- well, 12-well, 24-well, 48-well, 96-well, 384-well tissue culture dish.

12. The fluorescent reporter system of any one of claims 7-13, wherein the cell further comprises:

(b) a SARS-CoV-2 3CL^pro polypeptide.

13. The fluorescent reporter system of claim 12, wherein the 3CL^pro polypeptide comprises SEQ ID NO: 19, 20, 21, 22 or 23 or a polypeptide having at least 90% similarity to SEQ ID NO: 19-23.

14. The fluorescent reporter system of any one of claims 12 or 13, wherein the 3CL^pro polypeptide is encoded by a polynucleotide comprising SEQ ID NO: 24-28.

15. A method of detecting SARS-CoV-2 in a sample, the method comprising:

(a) contacting a sample comprising cells with the reporter of any one of claims 1 -

6, and

(b) detecting fluorescence in the cells, wherein fluorescence is indicative of SARS- CoV-2 infection.

16. The method of claim 15, wherein the sample is contacted with the cell for 8 hours or more.

17. The method of claim 15 or 16, wherein the detecting comprises quantifying the amount of fluorescence correlates with the amount of virus present in the sample.

18. A method of detecting SARS-CoV-2 in a sample, the method comprising:

(a) contacting the sample with the fluorescent reporter system of any one of claims 7-14, and

(b) detecting fluorescence in the reporter system, wherein fluorescence is indicative of SARS CoV-2 in the sample.

19. The method of claim 18, wherein the sample is contacted with the cell for 8 hours or more.

20. The method of claim 18 or 19, wherein the detecting comprises quantifying the amount of fluorescence correlates with the amount of virus present in the sample.

21. The method of any one of claims 15-20, wherein the sample is a biological sample from a subject having or suspected of having a SARS-CoV-2 infection.

22. The method of claim 21, wherein the sample is a nasal swab sample, a throat swab sample, a saliva sample, or a blood sample.

23. A method of screening for an agent capable of inhibiting SARS-CoV-23CL^pro , the method comprising:

(a) incubating the reporter system of claim 14 with the agent; and

(b) detecting the reduction in fluorescence in the system, wherein a reduction in fluorescent demonstrates the agents ability to block viral proteolytic cleavage by 3CL^pro.

24. The method of claim 23, wherein the method comprises (a) incubating the reporter system with different amounts of the agent; and (b) quantifying the fluorescence associated with the different amounts, wherein the fluorescence correlates with the IC50 for the agent.