Protocol for in vitro fluorescence assay of papain-like protease and cell-based immunofluorescence assay of coronavirus infection

Summary

Here, we describe detailed steps to constitute an in vitro assay for monitoring papain-like protease of coronavirus and a cell-based immunofluorescence infection assay. These assays can be adapted for high-throughput screening to determine the efficacy of novel protease inhibitors of coronaviruses and other viruses. In addition, cell-based immunofluorescence infection assay can be used to visually analyze antiviral efficacy of any novel compounds.

For complete details on the use and execution of this protocol, please refer to Jeong et al. (2022).¹

Graphical abstract

Highlights

• •In vitro assay that can be used for the characterization of viral proteases
• •Identification of protease inhibitors
• •Cell-based immunofluorescence assay to monitor viral infection
• •Characterization of compounds that inhibit viral infection

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we describe detailed steps to constitute an in vitro assay for monitoring papain-like protease of coronavirus and a cell-based immunofluorescence infection assay. These assays can be adapted for high-throughput screening to determine the efficacy of novel protease inhibitors of coronaviruses and other viruses. In addition, cell-based immunofluorescence infection assay can be used to visually analyze antiviral efficacy of any novel compounds.

Before you begin

Papain-like protease (PLpro) is a conserved protease of coronaviruses and plays an essential role in processing newly synthesized viral polyproteins along with the 3C-like protease (3CLpro). However, unlike the 3CLpro, PLpro has a deubiquitinase domain that prevents early stage of NF-κB signaling activation thereby silencing the innate antiviral immunity. Thus, inhibition of PLpro is an efficient way to limit the proliferation of coronaviruses. Since the PLpro of coronaviruses is structurally similar to each other, a drug targeting PLpro can serve as a broad-spectrum antiviral. This protocol describes the specific steps to monitor the enzymatic activity of PLpro in vitro in the presence or absence of inhibitors. Specifically, our protocol allows for ready examination of efficacy of PLpro inhibitors using both in vitro and cell-based assays. The cell-based assays described here can be employed to examine infection of other viruses in various genetically modified cell lines.

Institutional permissions

This protocol requires the use of viral strains for which the experiments should be performed in a BSL-3 facility following relevant governmental and institutional guidelines.

Preparation of PLpro plasmids

The coding sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) PLpro (aa 1,484−1,802), severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro (aa 1,541−1,854), and SARS-CoV-2 PLpro (aa 746−1,060) were obtained from the polyprotein sequences (GenBank: KT029139.1, AAY60792.1, and YP_009725299.1, respectively). Codon-optimized genes (IDT) were cloned into the NdeI/XhoI multi-cloning sites of the plasmid pET-21a, and DNA sequences were verified.¹

Maintenance of cell lines

Timing: 1–2 weeks

• 1.
  Maintenance of Vero E6 and Vero cells.

  • a.
    Maintain cell lines in Dulbecco′s Modified Eagle′s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic.

    Note: A fully confluent monolayer of Vero E6 and Vero cells usually contains approximately 2 × 10⁷ cells in a T175 flask.

  • b.
    Grow cells in a humidified 37°C, 5% CO₂ incubator.
  • c.
    Subculture cells using Trypsin-EDTA.

Viral propagation and titration

Timing: 1–2 weeks

• 2.
  Preparation of Vero E6 cells for viral propagation.

  • a.
    Collect the cells by trypsinization.
  • b.
    Centrifuge the cells at 450 × g for 5 min.
  • c.
    Wash the cells with DMEM containing 10% FBS without antibiotics.
  • d.
    Centrifuge at 450 × g for 5 min.
  • e.
    Repeat steps 2.c-d twice to remove the residual antibiotics.
  • f.
    Resuspend the cell pellet in DMEM containing 10% FBS without antibiotics.
  • g.
    Seed 1 × 10⁷ cells in a T175 flask in 30 mL of media.
  • h.
    Incubate the cells in a humidified 37°C, 5% CO₂ incubator for 24 h.

CRITICAL: The following steps need to be performed in a BSL-3 facility inside a biosafety cabinet. Please follow the appropriate procedure for entry into the BSL-3 facility.^2,3

• 3.
  Viral stock propagation.^4,5

  • a.
    Freshly thaw a vial of MERS-CoV / SARS-CoV / SARS-CoV-2 viral stock (stored at −130°C deep freezer).

    Note: Information on the viral strains used in this protocol is provided in the key resources table. However, the viral strains can be obtained from other sources where available.

  • b.
    Dilute the viral stock with 5 mL of pre-warmed DMEM containing 10% FBS.

    Note: The media used for viral propagation and titration is media containing only 10% FBS without antibiotics.

  • c.
    Remove media from cells prepared in step 2.h and dispense the virus prepared in step 3.b.
  • d.
    Absorb virus on a shaking rocker (speed of 50 rpm) at 23°C–25°C for 1 h.
  • e.
    Remove the virus inoculum and wash cells three times with Dulbecco’s phosphate-buffered saline (DPBS).
  • f.
    Add 15 mL of fresh DMEM containing 10% FBS and incubate the flask in a humidified 37°C, 5% CO₂ incubator for 72 h or until the cytopathic effect is observed.
  • g.
    Collect the supernatant from the infected Vero E6 cells and centrifuge at 900 × g for 10 min at 4°C.
  • h.
    Transfer the supernatant into a fresh tube and make 1 mL aliquots of the supernatant in 2 mL cryovial tubes.

    Note: Usually aliquoted into 10–15 tubes.

    Pause point: If not proceeding to step 4 right away, all propagated viral stocks can be kept in −130°C deep freezer until ready to proceed. When ready for use, thaw the stock at 23°C–25°C right before use. If proceeding to step 4 right away, one stock can be used for titration and the rest can be stored in a deep freezer.

• 4.
  Viral titration using plaque assay.⁶

  • a.
    Prepare Vero cells for plaque assay.

    • i.
      Seed 1 × 10⁶ Vero cells in 2 mL of media per well in 6-well plates.
    • ii.
      Incubate 16–18 h in a humidified 37°C, 5% CO₂ incubator.
  • b.
    Prepare dilutions of viral stock.

    • i.
      Prepare 10-fold serial dilutions of the propagated viral stock obtained from step 3.h at a range of 10⁻² to 10⁻⁸ using DMEM containing 1% FBS.
  • c.
    Remove culture medium from the confluent Vero cell monolayer from step 4.a.ii.
  • d.
    Wash the cells with DPBS and add 500 μL of dilutions (from step 4.b.i) in each well of a 6-well plate.

    Note: The first well of each plate is a mock control in which 500 μL of DMEM containing 1% FBS is placed without virus addition. Perform the assay in triplicates for more exact titration.

  • e.
    Adsorb virus on a shaking rocker (speed of 50 rpm) at 23°C–25°C for 1 h.
  • f.
    During the virus adsorption, prepare the overlay medium (materials and equipment) in a 50 mL conical tube.

    Note: Follow below procedure to make overlay medium (prepare overlay medium right before pouring into the wells):

    • i.
      Melt down the 1.3% bacto agar by microwaving.
    • ii.
      Keep stirring the agar and cool down the agar for about 20 min at 45°C.
    • iii.
      When agar has cooled down, add 2× DMEM (kept at 37°C) and FBS.
    • iv.
      Mix well.
  • g.
    Remove the virus inoculum after 1 h of virus adsorption and wash the infected cells with DPBS.
  • h.
    Immediately add 2 mL of overlay medium to each well.
  • i.
    Let the plates sit in the biosafety cabinet for 20 min as the overlay medium turns solid.
  • j.
    When the overlay medium becomes solid, move the plates to a humidified 37°C, 5% CO₂ incubator, and incubate for 3–4 days.

    Note: Incubate the plates upside down to prevent the evaporation of overlay medium and to prevent contamination due to water condensation on the plate lid.

  • k.
    After 3–4 days of incubation, check if there are visible plaques.
  • l.
    Add 1 mL of 4% paraformaldehyde to each well and fix the cells at 23°C–25°C for 30 min on a shaking rocker (speed of 50 rpm).
  • m.
    Carefully remove the overlay medium with spatula and discard.
  • n.
    Add 500 μL of 0.1% crystal violet solution (materials and equipment) to each well, and let the crystal violet solution spread on the shaking rocker (speed of 50 rpm) at 23°C–25°C for 10 min.
  • o.
    Wash the wells with water.
  • p.
    Count the number of plaques of the wells with around 20–300 plaques (Figure 1).

    Note: Viral titer can be calculated as below (Figure 1):

    $\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{q}\mathrm{u}\mathrm{e}\mathrm{f}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{t}\left(\raisebox{1ex}{$\mathrm{P}\mathrm{F}\mathrm{U}$}\!\left/ \!\raisebox{-1ex}{$\mathrm{m}\mathrm{L}$}\right.\right)=\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{q}\mathrm{u}\mathrm{e}\#\times \mathrm{d}\mathrm{i}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\times \frac{1\mathrm{m}\mathrm{L}}{\mathrm{i}\mathrm{n}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}}$

    For example, if average of 22 plaques appear in triplicate wells infected with 10⁻⁶ diluted virus, then

    $22\left(\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{q}\mathrm{u}\mathrm{e}\#\right)\times {10}^6\left(\mathrm{d}\mathrm{i}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\right)\times \frac{1\mathrm{m}\mathrm{L}}{0.5\mathrm{m}\mathrm{L}\left(\mathrm{i}\mathrm{n}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\right)}=\mathbf{4.4}\times {\mathbf{10}}^{\mathbf{7}}\raisebox{1ex}{$\mathbf{P}\mathbf{F}\mathbf{U}$}\!\left/ \!\raisebox{-1ex}{$\mathbf{m}\mathbf{L}$}\right.$

Figure 1.

Plaque assay is used to determine viral titer

For the current assay, wells that contain viral stock diluted at dilution factors of 10⁻⁶ and 10⁻⁵ contain countable plaque numbers (22 plaques at 10⁻⁶ and 248 plaques at 10⁻⁵ dilution). This confirms that the dilution was performed properly. The viral plaque-forming unit (PFU) can be calculated as below:$22\left(\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e}\#\mathrm{i}\mathrm{f}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{f}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{u}\mathrm{p}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{p}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{e}\right)\times {10}^6\left(\mathrm{d}\mathrm{i}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\right)\times \left(\frac{1\mathrm{m}\mathrm{L}}{500\mu \mathrm{L}\left(\mathrm{a}\mathrm{s}\mathrm{s}\mathrm{a}\mathrm{y}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\right)}\right)=\mathbf{4.4}\times {\mathbf{10}}^{\mathbf{7}}\raisebox{1ex}{$\mathbf{P}\mathbf{F}\mathbf{U}$}\!\left/ \!\raisebox{-1ex}{$\mathbf{m}\mathbf{L}$}\right.$.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit anti-MERS-CoV spike antibody (dilution: 0.25 μg/mL in 5% normal goat serum)	Sino Biological	Cat# 40069-R723; RRID: AB_2860455
Mouse anti-SARS-CoV spike S1 antibody (dilution:0.25 μg/mL in 5% normal goat serum)	Sino Biological	Cat# 40150-MM02; RRID: AB_2860459
Rabbit anti-SARS-CoV-2 protein antibody (dilution: 0.25 μg/mL in 5% normal goat serum)	Sino Biological	Cat# 40143-T62; RRID: AB_2892769
Goat anti-Mouse IgG (H + L) Alexa Fluor™ 488 (dilution: 0.5 μg/mL in 5% normal goat serum)	Invitrogen Thermo Fisher Scientific	Cat# A-11029 RRID: AB_2534088
Goat anti-Rabbit IgG (H + L) Alexa Fluor™ 488 (dilution: 0.5 μg/mL in 5% normal goat serum)	Invitrogen Thermo Fisher Scientific	Cat# A-11008 RRID: AB_143165
Bacterial and virus strains
MERS-CoV	Korea National Institute of Health (KNIH)	KOR/KNIH/2015
SARS-CoV	Laboratory of Prof. JSM Peiris (University of Hong Kong)	HK39849
SARS-CoV-2	Korea Centers for Disease Control and Prevention	BetaCov/Korea/KCDC03/2020
Chemicals, peptides, and recombinant proteins
Lysozyme	Goldbio	Cat# L-040-10
Crystal violet	Sigma-Aldrich	Cat# C0775
Hoechst 33342	Invitrogen Thermo Fisher Scientific	Cat# H-3570
RLRGG-AMC peptide	Bachem	Cat# I-1690.0025
6-Thioguanine	Sigma-Aldrich	Cat# A4882
Ni-NTA agarose beads	Qiagen	Cat# 1018244
Experimental models: Cell lines
Vero cells	American Type Culture Collection	Cat# CCL-81; RRID: CVCL_0059
Vero E6 cells	American Type Culture Collection	Cat# CRL-1586 RRID: CVCL_0574
Software and algorithms
Operetta CLS High-Content Analysis system	PerkinElmer	https://www.perkinelmer.com/de/category/operetta-cls-high-content-analysis-system
Harmony High-Content Imaging and Analysis software	PerkinElmer	https://www.perkinelmer.com/product/harmony-4-9-office-license-hh17000010
Columbus Image Data Storage and Analysis system™	PerkinElmer	https://www.perkinelmer.com/product/image-data-storage-and-analysis-system-columbus
GraphPad Prism® software	GraphPad Software	https://www.graphpad.com/
Other
Dulbecco’s Modified Eagle’s Medium (DMEM)	Welgene	Cat# LM 001-07
DMEM, powder	Gibco Thermo Fisher Scientific	Cat# 12800017
Opti-PRO™ SFM	Gibco Thermo Fisher Scientific	Cat# GIB-12309-019
Antibiotic-antimycotic	Gibco Thermo Fisher Scientific	Cat# GIB-15240-062
Dulbecco’s phosphate-buffered saline (D-PBS) (1×), liquid	Welgene	Cat# LB 001-02
BL21 competent E. coli cell stock	New England Biolabs	Cat# C2530H
Sealing film	Axygen	Cat# PCR-SP
μ-clear F-bottom black plates	Greiner	Greiner CELLSTAR® microplate 781090
Protein purification column	Bio-Rad	Poly-prep® chromatography column 7311550
Protein concentration filter	Millipore	Amicon® ultra-15 centrifugal filter unit UFC901024
Buffer exchange membrane	Thermo Scientific	Slide-A-Lyzer dialysis cassette 66370
Cell sonicator	Sonics & Materials	Ultrasonic processor VCX750-220V
Nanodrop	Implen	NanoPhotometer® N60
Plate reader	BioTek	Cytation5 imaging reader
Automated liquid handler	PerkinElmer	Flexible JANUS® G3 automated workstation AJV001
High throughput screening station	PerkinElmer	Multimodule ultra-high throughput screening platform

Materials and equipment

Overlay medium

Reagent	Final concentration	Amount
2× DMEM∗	1× DMEM	24.5 mL
1.3% bacto agar	0.65% bacto agar	25 mL
FBS	1% (v/v)	0.5 mL
Total	N/A	50 mL

Note: Prepare 1.3% bacto agar in double distilled water (ddH₂O), autoclave, and store at 23°C–25°C until ready for use (agar will solidify). On the day of experiment, follow the procedure under step 4.f in the Before You Begin section to prepare overlay medium.

∗2× DMEM

Reagent	Final concentration	Amount
Powdered DMEM	N/A	1 pk
Sodium bicarbonate	88 mM	3.70 g
HEPES	40 mM	4.77 g
Total (ddH2O)	N/A	500 mL

Note: It is necessary to mix 2× DMEM well and filter using 0.22 μm PVDF membrane filter.

0.1% crystal violet solution

Reagent	Final concentration	Amount
4% crystal violet solution∗	0.1% crystal violet	25 mL
1× phosphate-buffered saline (PBS)	N/A	975 mL
Total	N/A	1000 mL

∗4% crystal violet solution

Reagent	Final concentration	Amount
Powdered crystal violet	4% (w/v)	4 g
Total (absolute ethanol)	N/A	100 mL

DNase stock

Reagent	Final concentration (in ddH2O)
DNase	100 μg/mL
CaCl2	5 mM

Lysis buffer

Reagent	Final concentration (in ddH2O)
Trizma base	20 mM
NaCl	250 mM
Glycerol	5% (v/v)
Triton X-100	0.2% (v/v)
β-mercaptoethanol	2 mM
pH	8.5

Wash buffer

Reagent	Final concentration (in ddH2O)
Trizma base	20 mM
NaCl	250 mM
Imidazole	8 mM
β-mercaptoethanol	2 mM
pH	8.5

Elution buffer

Reagent	Final concentration (in ddH2O)
Trizma base	20 mM
NaCl	30 mM
Imidazole	150 mM
β-mercaptoethanol	2 mM
pH	8.5

Assay buffer for PLpro enzyme assay

Reagent	Final concentration (in ddH2O)
HEPES	50 mM
Triton X-100	0.01% (v/v)
BSA	0.1 mg/mL
Dithiothreitol (DTT)	5 mM
pH	7.5

Alternatives: 2 mM reduced L-glutathione can be used instead of 5 mM DTT.

Coomassie blue staining solution

Reagent	Final concentration
Coomassie brilliant blue	0.2% (w/v)
Methanol	50% (v/v)
Glacial acetic acid	10% (v/v)
ddH20	40% (v/v)

Note: Stir the solution for 3–4 h and then filter through Whatman filter paper grade 1.

CRITICAL: Take caution when dealing with glacial acetic acid (strong acid). Pour inside the chemical hood to avoid fumes.

Coomassie blue de-staining solution

Reagent	Final concentration
Methanol	40% (v/v)
Glacial acetic acid	10% (v/v)
ddH20	50% (v/v)

FPLC buffer

Reagent	Final concentration (in ddH2O)
HEPES	50 mM
NaCl	0.5 M
pH	7.5

Step-by-step method details

Purification of PLpro protein

Timing: 4–5 days

This step outlines steps needed to be taken for purification of PLpro protein. The step is subdivided into 3 main steps: (1) transformation of PLpro-expressing plasmid into competent cells, (2) preparation and lysis of PLpro-expressing cells for protein purification, and (3) protein purification.

• 1.
  Transformation of PLpro plasmid into competent cells.

  Note: Turn on the water bath to keep the temperature at 42°C.

  • a.
    Obtain BL21 competent cell stock (materials and equipment) and keep on ice for 20 min.
  • b.
    Place 50 μL of BL21 stock into a new pre-chilled 1.6 mL Eppendorf tube.
  • c.
    Mix cells from step 1.b with 3–5 μL of plasmid and mix by tapping.
  • d.
    Place the mixture on ice for 30 min.
  • e.
    Place the tube in the water bath kept at 42°C for 40 s.
  • f.
    Move the reaction to ice for 2 min.
  • g.
    Add 300 μL LB media and incubate at 37°C with shaking at 200 rpm for 1 h.
  • h.
    Spread the bacterial mixture on LB plates containing 100 μg/mL carbenicillin.

    Note: Spread different volumes (i.e., 50–150 μL) of ligation mixture to avoid having plates with too many colonies.

  • i.
    Place the plates 16–18 h in a 37°C incubator until colonies appear.
  • j.
    If proceeding to step 2 right away, inoculate a single colony in 3 mL liquid LB media containing 100 μg/mL carbenicillin in 14-mL plastic round-bottom culture tubes.

    Note: Make bacterial stocks (25% glycerol) with colonies for future use.

  • k.
    Incubate step 1.j 16–18 h at 37°C with shaking at 250 rpm.

    Pause point: If not proceeding to step 2 right away, the stocks made in step 1.j can be kept at −80°C until ready to proceed. When ready to proceed to step 2, streak the bacterial stock on LB plates containing 100 μg/mL carbenicillin and incubate 16–18 h at 37°C. Then proceed from step 1.j.

• 2.
  Growing of bacterial cells expressing PLpro.

  • a.
    Prepare fresh liquid LB media containing 100 μg/mL carbenicillin.

    Note: The volume of media depends on how much protein is needed.

    Culture volumea	Amount of protein	Yield (mg per L culture)
    200 mL	2 mg	10 mg/L
    500 mL	4 mg	8 mg/L
    1.5 L	15 mg	10 mg/L

    ^a

    Culture at a volume of 200 and 500 mL is grown in a 1 L and 2 L flask, respectively, while culture at a volume of 1.5 L is grown in a 5 L flask.
  • b.
    Measure OD₆₀₀ of the culture from step 1.k.
  • c.
    Re-inoculate in LB media prepared in step 2.a at OD₆₀₀ = 0.01.
  • d.
    Incubate step 2.c at 37°C with shaking (at 170 RPM) for 3–5 h until OD₆₀₀ reaches 0.6–0.7.

    CRITICAL: Prepare 1 M IPTG (made in ddH₂O and filtered through 0.2 μm filter) right before proceeding to step 2.e. Be sure to not let OD₆₀₀ of the culture exceed 0.7.

  • e.
    Add IPTG at a final concentration of 0.5 mM into step 2.d.
  • f.
    Incubate at 20°C for 14–18 h (shaking at 150 RPM).
  • g.
    Centrifuge the culture at 6,000 × g for 15 min at 4°C.
  • h.
    When the centrifugation is over, pour out the supernatant and proceed to step 3.

    Pause point: If not proceeding to step 3 right away, the cells obtained in step 2.h can be kept at −80°C until ready to use.

• 3.
  PLpro protein purification.

  • a.
    Add appropriate volume of lysis buffer (materials and equipment) to the cells.

    Note: Lysis buffer needs to be stored at 4°C and can be used up to 6 months. The volume of lysis buffer depends on culture volume.

    Culture volume	Volume of lysis buffer
    200 mL	10 mL
    500 mL	10 mL
    1.5 L	30–40 mL

  • b.
    Add DNase stock (materials and equipment) and MgCl₂ stock (1 M stock prepared in water) at a final concentration of 100 μg/mL and 10 mM, respectively.

    Note: DNase stocks need to be stored at −20°C after preparation and can be used up to 3 months, and 1 M MgCl₂ stock can be prepared earlier and kept at 23°C–25°C.

  • c.
    Prepare lysozyme stock at 100 mg/mL (in ddH₂O) and add at a final concentration of 100 μg/mL to step 3.b.

    Note: Prepare the lysozyme stock fresh every time before use.

  • d.
    Incubate 30 min while mixing every 10 min.
  • e.
    Sonicate to lyse the bacterial cells with cell sonicator (materials and equipment).
    Setting to [1 s ON + 1 s OFF; repeat 45×].

    Note: The power output of the sonicator (Sonics & Materials VCX750-220V) is 750 Watt 20 kHz, and the amplitude has been set to 60% for the lysis procedure.

    CRITICAL: Prepare ice. When one cycle of step 3.e is over, place the samples on ice for 1–2 min to cool down before proceeding to another cycle.

  • f.
    Repeat 5–6 or 10–12 cycles of [1 s ON + 1 s OFF; repeat 45×] for smaller or larger volumes, respectively, until the lysis is complete (turns brown).
  • g.
    Centrifuge the lysis mixture at 4°C at 16,000 × g for 30 min.
  • h.
    During centrifugation, prepare columns with Ni-NTA beads:

    • i.
      Add appropriate volume of Ni-NTA beads to the protein purification columns (materials and equipment).

      Note: The volume of beads depends on culture volume.

      Culture volume	Volume of Ni-NTA beads
      200 mL	200 μL
      500 mL	500 μL
      1.5 L	2 mL

    • ii.
      Add 10 mL of ddH₂O and let it run through the column.
    • iii.
      Add 10 mL of lysis buffer and let it run through the column.
    • iv.
      Repeat step 3.h.iii twice.
    • v.
      Close the bottom cap of the column.

      CRITICAL: The cap needs to be tightly closed so that the samples do not run out when proceeding to step 3.i.

  • i.
    Add the supernatant from centrifugation of step 3.g to the column (with cap closed) and mix well with the beads.

    Note: Keep ∼50 μL of centrifuged lysate for SDS-PAGE in step 3.q.

  • j.
    Incubate the cell lysate with the beads at 4°C in a circular shaker for 1.5–2 h to allow binding of protein to the beads.
  • k.
    When the incubation is over, let the columns stand upright for 10 min until beads (now bound to protein) settle to the bottom.
  • l.
    Remove the cap in the bottom of the column and let the lysate (now containing no protein) flow out of the columns.

    Note: Keep ∼50 μL of the filtrate for SDS-PAGE in step 3.q. Filtrate should contain proteins but not the target protein.

  • m.
    Wash the beads with 10 mL of wash buffer (materials and equipment) by letting the wash buffer run through the column.

    Note: Wash buffer needs to be stored at 4°C and can be used up to 6 months. Collect ∼50 μL of the first wash for SDS-PAGE in step 3.q. Wash should contain minimal amount of protein since protein should be well-bound to the beads.

  • n.
    Repeat step 3.m twice.
  • o.
    Elute the protein using 300–500 μL of elution buffer (materials and equipment).

    Note: Elution buffer needs to be stored at 4°C and can be used up to 6 months.

  • p.
    Repeat step 3.o 10 times and collect the eluate.
  • q.
    Run the samples collected from step 3.i, step 3.l, step 3.m, and step 3.p on a SDS-PAGE gel to check for presence of protein.

    Note: Mix ∼10 μL of samples with loading buffer for SDS-PAGE.

  • r.
    Stain the gel using Coomassie blue staining solution (materials and equipment) at 23°C–25°C for 1–2 h with rocking (speed of 100 rpm).
  • s.
    Wash the gel using Coomassie blue de-staining solution (materials and equipment) for 16–18 h at 23°C–25°C with rocking (speed of 100 rpm)
  • t.
    Determine which eluates contain PLpro protein by analyzing the gel image (Figure 2).
  • u.
    Combine the protein-containing eluates and measure the protein concentration using a nanodrop instrument.
  • v.
    Use protein concentration filter (materials and equipment) to concentrate the sample to an ideal volume for FPLC.

    Note: Follow the link (https://images-na.ssl-images-amazon.com/images/I/71A8g+u8BrL.pdf) for the manufacturer’s instructions.

    Note: The injection volume for the FPLC column used for the current protocol is 500 μL. Considering the dead volume during injection, the samples have been concentrated to 600-700 μL.

  • w.
    Purify the protein sample using FPLC.
    FPLC buffer

    Reagent	Final concentration
    HEPES	50 mM
    NaCl	0.5 M
    pH	7.5

    Note: Buffer needs to be filtered and stored at 4°C and can be used up to 3 months; ∼500 mL buffer is needed to run one sample.

    FPLC column
    Superdex 200 10/300 GL column (GE Healthcare) or equivalent (e.g., Superose 6 Increase 10/300 GL Column).
    FPLC setting
    90 eluates (400 μL each); flow rate = 0.4 mL/min.

    Note: Centrifuge the samples at 10,000 × g for 10 min to purify them before injecting into FPLC.

    Alternatives: Alternative to FPLC, buffer exchange membrane (Materials and Equipment) can be used. Follow the link (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011337_SlideALyzer_Dialy_Cass_UG.pdf) for the manufacturer’s instructions.

  • x.
    Confirm which FPLC eluates contain pure protein by analyzing generated FPLC peak (Figure 3).
  • y.
    Combine the eluates containing the protein.
  • z.
    Measure the concentration of the combined eluates from step 3.y using a nanodrop instrument.
  • aa.
    Prepare the sample stocks in 10% glycerol (Add 50% glycerol at 1/5 of the total volume).

    Note: An ideal final stock concentration is 4–5 mg/mL. Concentrate step 3.y if the protein concentration is too low using protein concentration filter (materials and equipment). Follow the link (https://images-na.ssl-images-amazon.com/images/I/71A8g+u8BrL.pdf) for the manufacturer’s instructions.

Figure 2.

SDS page confirms eluates containing protein

For the current assay, eluate #1, eluate #2, and eluate #3 contain the target protein therefore can be combined and used for the measurement of protein concentration.

Figure 3.

FPLC purifies the protein

For the current protocol, FPLC fraction is collected at 400 μL each. Fractions 43–53 generated a protein peak and thereby can be combined as a sample.

Fluorescence-based PLpro enzyme assay

Timing: 1 day

This step outlines steps required to perform the fluorescence-based PLpro enzyme assay.^7,8,9 The assay utilizes a short peptide-linked AMC (7-amido-4-methylcoumarin), specifically with a sequence R-L-R-G-G-AMC, which is cleaved into AMC upon recognition by PLpro. When PLpro cleaves AMC peptide to produce AMC, a fluorescence signal is generated and can be measured (Figure 4). First, checkerboard assay analysis is used to determine the optimal enzyme (PLpro) and substrate (peptide-linked AMC) concentration. Then, the determined optimal concentration is used to perform the enzyme assay in a 384-well plate format. This protocol can be adjusted for the use of other proteins by changing the sequence of the substrate.

• 4.
  Determination of optimal enzyme and substrate concentrations using checkerboard assay.

  • a.
    Prepare the AMC substrate at a concentration of 400 μM in assay buffer (materials and equipment).

    Note: Assay buffer needs to be stored at 4°C and can be used up to 6 months.

  • b.
    Prepare the PLpro protein at a concentration of 20 μM in assay buffer.
  • c.
    Prepare the plate for the checkerboard assay as follows:

    • i.
      Add 40 μL of assay buffer to columns 2–12, rows A-L of a 384-well plate (blue circles in Figure 5A).
    • ii.
      Place 80 μL of the 400 μM AMC substrate prepared in step 4.a into column 1, row A (red circle in Figure 5A).
    • iii.
      Dilute 400 μM AMC substrate into half in assay buffer to make 200 μM AMC substrate.
    • iv.
      Place 80 μL of the 200 μM AMC substrate to column 1, rows B-L (green circles in Figure 5A).
    • v.
      Move 40 μL of contents in column 1 to column 2 using multi-channel pipette. Then move 40 μL of contents in column 2 to column 3 and so on until reaching column 11 (blue arrows in Figure 5A).

      Note: Mix thoroughly and change tips before moving to the next dilution.

    • vi.
      When column 11 is reached, discard 40 μL of the content.

      Note: Column 12 will contain no AMC substrate.

    • vii.
      Place 40 μL of 20 μM PLpro prepared in step 4.b into all wells of row A (yellow square in Figure 5A).
    • viii.
      Move 40 μL of contents from row A to row B using multi-channel pipette. Then move 40 μL of contents from row B to row C and so on until reaching row K (red arrows in Figure 5A).

      Note: Mix thoroughly and change tips before moving to the next dilution.

    • ix.
      When row K is reached, discard 40 μL of the content.

      Note: Row L will contain no PLpro enzyme. In the end, the highest AMC substrate concentration is 200 μM, and the highest enzyme concentration is 10 μM.

    • x.
      Incubate the plate for 1 h at 23°C–25°C protected from light.
    • xi.
      Measure fluorescence at 360 nm (excitation) / 450 nm (emission) using a plate reader (materials and equipment) (troubleshooting 1 and 2).
    • xii.
      Determine the optimal enzyme and substrate concentration that yields maximal un-saturated signal (Figure 5B).

      Alternatives: The procedures are written for 384 well format. However, it can be adjusted for 96 well format by increasing the total assay volume to 150 μL instead of 40 μL (adjust all volumes proportionally).

• 5.
  Performing PLpro enzyme assay.

  • a.
    Prepare the AMC substrate in an assay buffer at a concentration 4 times the optimal concentration determined in step 4.
  • b.
    Prepare the PLpro enzyme in an assay buffer at a concentration 4 times the optimal concentration determined in step 4.
  • c.
    Prepare the compounds of interest at 2 times the target compound concentration.
  • d.
    Add 10 μL of AMC substrate prepared in step 5.a into needed wells.
  • e.
    Add 10 μL of PLpro enzyme prepared in step 5.b into needed wells.
  • f.
    Add 20 μL of compounds prepared in step 5.c into needed wells.
  • g.
    Incubate the plate for 1 h at 23°C–25°C protected from light.
  • h.
    Measure fluorescence at 360 nm (excitation) / 450 nm (emission) using a plate reader.

Note: To validate the assay, include wells with 6-Thioguanine, a known PLpro inhibitor. Be sure to include positive and negative controls, which are wells with maximal and minimal fluorescence signal, respectively. Positive control will be wells with AMC substrate and enzyme without any compounds, while wells with only AMC substrate serve as negative control. Include the negative and positive controls in at least triplicates. The values for positive and negative controls are used to calculate the Z′ factor

${\mathrm{Z}}^{\prime }=1-\frac{3\left(\sigma \mathrm{p}+\sigma \mathrm{n}\right)}{\left|\mu \mathrm{p}-\mu \mathrm{n}\right|}$

where μ and σ are the average and standard deviation, respectively, of the signals of positive (p) and negative (n) control of the assay plates. Also, include 6-Thioguanine as a known PLpro inhibitor for validation of the assay condition.

Figure 4.

Short peptide-linked AMC produces fluorescence signal upon being cleaved by PLpro

When a short peptide-linked AMC (7-amido-4-methylcoumarin), specifically with a sequence R-L-R-G-G-AMC, is cleaved into AMC upon recognition by PLpro, the resulting fluorescence can be measured at 360 nm (excitation wavelength) / 450 nm (emission wavelength). The efficacy of compound against PLpro can be measured by the fluorescence signal.

Figure 5.

Checkerboard assay is used to determine optimal PLpro and short peptide-linked AMC concentrations

(A) A 384-well plate can be designed as outlined.

(B) Maximal concentrations of PLpro and peptide-linked AMC that produce linear relationship without producing saturated signals are chosen as optimal. In this current assay, PLpro concentration of 2.5 μM and substrate (peptide-linked AMC) concentration of 40–50 μM can be chosen as optimal. RFU: relative fluorescence unit. This figure is reproduced from Supplementary Figure 1A of Jeong et al.¹

Applications of PLpro enzyme assay

Timing: 1–2 days

The fluorescence-based enzyme assay can be applied for 1) determination of IC₅₀ of the test compounds or 2) high-throughput screening.

• 6.
  Application 1: Determination of IC₅₀ of the test compounds.

  • a.
    Prepare the AMC substrate in an assay buffer at a concentration 4 times the optimal concentration determined in step 4.
  • b.
    Prepare the PLpro enzyme in an assay buffer at a concentration 4 times the optimal concentration determined in step 4.
  • c.
    Prepare dilutions of the test compound. Be sure to prepare the highest concentration at 2 times the target final compound concentration.
  • d.
    Add 10 μL of AMC substrate prepared in step 6.a into needed wells.
  • e.
    Add 10 μL of PLpro enzyme prepared in step 6.b into needed wells.
  • f.
    Add 20 μL of compound dilutions prepared in step 6.c into needed wells.

    Note: Similar to checkerboard assay, this protocol can be adjusted for 96 well plates by increasing the total assay volume to 150 μL instead of 40 μL (adjust all volumes proportionally). Perform the assay in at least duplicates. Also, 6-Thioguanine can be included as a known PLpro inhibitor for validation of the assay condition.

  • g.
    Incubate the plate for 1 h at 23°C–25°C protected from light.
  • h.
    Measure fluorescence at 360 nm (excitation) / 450 nm (emission) using a plate reader.
  • i.
    Once the data is generated (Figure 6), calculate IC₅₀ using nonlinear regression with GraphPad Prism® software.

• 7.
  Application 2: High-throughput screening.

  • a.
    Prepare the AMC substrate in an assay buffer at a concentration 2 times the optimal concentration determined in step 4.
  • b.
    Prepare the PLpro enzyme in an assay buffer at a concentration 2 times the optimal concentration determined in step 4.
  • c.
    Add 20 μL of AMC substrate prepared in step 7.a into needed wells using an automated liquid handler (materials and equipment).
  • d.
    Add 20 μL of PLpro enzyme prepared in step 7.b into needed wells using an automated liquid handler.
  • e.
    Take the plates for addition of compounds using a high-throughput screening machine (materials and equipment).

    Note: Refer to Figure 7A for design of plate for high-throughput screening. Be sure to include negative and positive controls. Each plate can contain 320 compounds, such that 34 plates are sufficient for more than 10,000 compounds.

  • f.
    Incubate the plate for 1 h at 23°C–25°C away from light.
  • g.
    Measure fluorescence using the plate reader.
  • h.
    Once the data is generated, calculate Z′ factor for determination of assay quality and analyze the data for identification of hits (Figure 7B).

    Note:${\mathrm{Z}}^{\prime }=1-\frac{3\left(\sigma \mathrm{p}+\sigma \mathrm{n}\right)}{\left|\mu \mathrm{p}-\mu \mathrm{n}\right|}$

    μ and σ are the average and standard deviation, respectively, of the signals of positive (p) and negative (n) control.

    Z′ factor can be calculated for each plate, and the average of the Z′ factors becomes a Z′ factor for the assay. Z′ factor closer to 1 indicates good quality with small variation. As stated above, positive control will be wells with AMC substrate and enzyme without any compounds, while wells with only AMC substrate serve as negative control. Include the negative and positive controls in at least triplicates. Also, include 6-Thioguanine as a known PLpro inhibitor for validation of the assay condition.

Figure 6.

PLpro enzyme assay can be used to determine IC₅₀ of test compounds

A 384-well plate can be designed for determination of compound IC₅₀ using PLpro enzyme assay. The generated data can be used for determination of IC₅₀ values using software such as GraphPad Prism®. This figure is reproduced from Figure 1D of Jeong et al.¹

Figure 7.

PLpro enzyme assay can be used for high-throughput compound screening

(A) A 384-well plate can be designed for high-throughput screening as outlined.

(B) The resulting data can be plotted as % inhibition for each compound tested. In the current data, compounds that generated 0%–40% signal inhibition (marked with gray circles) were excluded; compounds that generated more than 40% signal inhibition (marked with red circles) were analyzed for hit identification. Signals from negative and positive controls for each plate were also plotted with blue and tan circles, respectively. This figure is reproduced from Figure 1C of Jeong et al.¹

Immunofluorescence-based virus infection assay

Timing: 1 week

The following steps describe how to perform cell-based immunofluorescence assay that can be effectively used to visualize viral infection in the host cells and analyze the antiviral efficacy of compounds by fluorescence microscopy.¹⁰

• 8.
  Preparation of Vero cells for viral infection.

  Note: Be sure to use μ-clear F-bottom black plates (materials and equipment). Refer to Figure 8A for possible plate design.

  • a.
    Collect the Vero cells by trypsinization from plates that are 80%–100% confluent.
  • b.
    Centrifuge for 3 min at 120 × g.
  • c.
    Resuspend the cells in complete Opti-PRO™ SFM (containing 1× Antibiotic-Antimycotic and 4 mM L-Glutamine).
  • d.
    Seed 1.2 × 10⁴ cells in each well of a 384-well plate in a media volume of 30 μL.
  • e.
    Incubate cells 16–18 h in a humidified 37°C, 5% CO₂ incubator.

• 9.
  Preparation and addition of compounds.

  • a.
    Prepare compounds at 5 times the target final concentration in complete Opti-PRO™ SFM.

    Note: As 10 μL of virus will be used for infection, the total reaction volume in a 384-well will be 50 μL (30 μL culture volume + 10 μL compound + 10 μL virus), resulting in a 5-fold dilution of compound concentrations. Also, be sure that the DMSO concentration does not exceed 5% so that the final DMSO concentration in the reaction will be less than 1%.

    CRITICAL: To validate the assay, include reference compounds with known antiviral activities.

  • b.
    Make dilutions that are appropriate for the purpose (i.e., serial dilution is standard).

    Note: Add DMSO to the diluting media to keep DMSO concentration same for all wells.

  • c.
    Add 10 μL of diluted compounds to the assigned wells.

    CRITICAL: The following steps need to be performed at BSL-3 facility inside a biosafety cabinet. Please follow the appropriate procedure for entry into the BSL-3 facility and for all following procedures.^2,3

• 10.
  Infection with virus.

  • a.
    Freshly thaw a vial of virus stored in a −130°C deep freezer inside a BSL-3 facility.
  • b.
    Reconstitute the coronavirus with complete Opti-PRO™ SFM to infect the cells with virus at target multiplicity of infection (MOI) in a volume of 10 μL:

    • i.
      MERS-CoV at an MOI of 0.0625.
    • ii.
      SARS-CoV at an MOI of 0.05.
    • iii.
      SARS-CoV-2 at an MOI of 0.08.

      Note: MOI (ratio of the # of viral particles to the # of target cells) can be calculated as below: $\mathrm{M}\mathrm{O}\mathrm{I}=\frac{\raisebox{1ex}{$\mathrm{P}\mathrm{F}\mathrm{U}$}\!\left/ \!\raisebox{-1ex}{$\mathrm{m}\mathrm{L}$}\right.\times \mathrm{i}\mathrm{n}\mathrm{o}\mathrm{c}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}}{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}$

      Note: Include wells with virus only or media only as infection and no infection controls, respectively.

  • c.
    Incubate the plate for 24 h in a humidified 37°C, 5% CO₂ incubator.

    Note: Do not stack the plates to decrease plate-to-plate variation due to the difference in incubation conditions for the plate placed on the top and for the plate placed on the bottom.

• 11.
  Immunofluorescence staining.

  • a.
    Dispense 17 μL of 16% paraformaldehyde solution to all wells to fix cells.

    Note: This will make final paraformaldehyde concentration to 4%.

  • b.
    Incubate at 23°C–25°C for 30 min.
  • c.
    Wash all wells twice with 80 μL of DPBS.
  • d.
    Dispense 25 μL of 0.25% Triton X-100 to each well to permeabilize cells.
  • e.
    Incubate at 23°C–25°C for 5 min.
  • f.
    Wash all wells twice with 80 μL of DPBS.
  • g.
    Dispense 25 μL of primary antibody (0.25 μg/mL in 5% normal goat serum).

    • i.
      Rabbit anti-MERS-CoV spike antibody (Sino Biological)
    • ii.
      Mouse anti-SARS-CoV spike S1 antibody (Sino Biological)
    • iii.
      Rabbit anti-SARS-CoV-2 nucleocapsid antibody (Sino Biological)
  • h.
    Incubate for 1 h at 37°C.
  • i.
    Wash all wells twice with 80 μL of DPBS per well.
  • j.
    Dispense 25 μL of secondary antibody (0.5 μg/mL in 5% normal goat serum) containing 1 μg/mL of Hoechst 33342.

    Note: Secondary antibody differs depending on the primary antibody.

  • k.
    Incubate for 1 h at 37°C protected from light.
  • l.
    Wash all wells twice with 80 μL of DPBS.
  • m.
    After washing, fill up the wells with 50 μL DPBS.
  • n.
    Seal the plate with sealing film and proceed to image acquisition using Operetta CLS High Content Analysis System controlled by software Harmony™.

    Note: Visit https://www.perkinelmer.com/de/Product/operetta-cls-system-hh16000000 for information on the software and equipment.

• 12.
  Image acquisition using HCS (high-content screening) imaging platform.

  • a.
    Place the plate in the HCS imaging platform and define the plate type.
  • b.
    Select the appropriate light source and filter.

    Note: Detection wavelength ranges from 365−465 nm for Hoechst 33342 and 495−518 nm for Alexa Fluor 488.

  • c.
    Choose “non-confocal” for operational mode and select the objective.
  • d.
    Adjust the focus and height of Z-stack using a well with infected Vero cells for each channel.
  • e.
    Confirm the brightness and select exposure time for each channel.
  • f.
    Test the selected parameters on one or two additional wells and adjust settings if necessary.
  • g.
    Select scan area and number of fields.
  • h.
    Start the image acquisition.

    Note: Choosing “all fields” in plate layout definition will prolong the time of measurement. Refer to Figure 8B for example images (troubleshooting 3).

• 13.
  Analysis of acquired images using Columbus Image Data Storage and Analysis System™.

  Note: For data transfer from the Operetta CLS High-Content Analysis System to Columbus, export TIF files and image-related data from the Harmony software and import data to Columbus. The following steps will provide details.

  • a.
    Open “Settings” and then “Data Management”, and “Export Data” in the Harmony software.
  • b.
    Specify “Columbus Export”.
  • c.
    Select data and measurements to be exported.
  • d.
    Specify “Export Path”.
  • e.
    Start the export from Harmony by clicking “Start”.
  • f.
    Open the Columbus software and move to the “Import” screen. The “Columbus Helper Required” window will appear.
  • g.
    Specify the “Import Type: Operetta IDX/TIF” and “Source Folder”.
  • h.
    Start the import to Columbus by clicking “Start”.
  • i.
    Open Columbus software.
  • j.
    Move to “Image Analysis” screen.
  • k.
    Select the measurement to analyze from the Data Tree.
  • l.
    Select one single image as representative image and the selected image is displayed in the Image View section.
  • m.
    Click “Load Analysis from Disk” and “Choose File”, and then open the ∗.aas file analysis script file.

    Note: The script file is created from predefined building blocks.

    The image analysis script does not generate data for single cell analysis by default. Therefore, make sure to include the following settings in the building block analysis script:
    In the Define Results building block, click on Object Results and specify to save ALL. Results per object are saved to the database.
    In addition, all object properties which are calculated by the building blocks (e.g., Calculate Intensity Properties, Calculate Morphology Properties) are also saved to the database.
    − Click on “save the analysis script” when finished creating the script file.
    − Run a Batch Analysis.
  • n.
    Click “Open” and “OK”.
  • o.
    Adjust and test the settings.

    • i.
      The segmentation of defined objects is displayed in the Image View. The illustration of the segmentation can be adjusted as preferred.
    • ii.
      Adjust the filters and input parameters of the building blocks to optimize the segmentation of objects.
    • iii.
      Define the final readout parameters of the analysis in the last building block “Define Results”.
    • iv.
      Select a few other image fields to test and optimize the analysis with these images.

      Note: The building blocks “Find Nuclei”, “Find Cytoplasm”, and “Select Population” require adjustment of the filter methods and parameters according to imaging settings, the cell type and density used, and the intensity of the dyes.

  • p.
    Save the analysis to the database or the file system as an ∗.aas file.
  • q.
    Move to “Batch Analysis” screen and select one or multiple measurements in the Data Tree.
  • r.
    Click "…" to load the saved analysis script file and then the green arrow to start the batch analysis.
  • s.
    Use the Job Status screen to view the progress of the analysis.
  • t.
    The results are reported to the database.
  • u.
    Move to “Export” screen and connect to Columbus Helper.
  • v.
    Select the measurement from the Data Tree.
  • w.
    Specify export options and choose a destination export folder on the server.
  • x.
    Select the type of file for the export and click the green arrow to start the export.
  • y.
    The results can be found as a Text Document in a previously defined folder.
  • z.
    Graphs for the quantification of infectivity (Figure 8C) can be used for calculation of various parameters (troubleshooting 4).

    Note:Percent inhibition (PI) is normalized as follows:

    $\mathrm{P}\mathrm{I}=\left[1-\frac{\left(\mathrm{I}\mathrm{N}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}–\mu \mathrm{I}\mathrm{N}\mathrm{m}\mathrm{o}\mathrm{c}\mathrm{k}\right)}{\left(\mu \mathrm{I}\mathrm{N}\mathrm{v}\mathrm{e}\mathrm{h}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}–\mu \mathrm{I}\mathrm{N}\mathrm{m}\mathrm{o}\mathrm{c}\mathrm{k}\right)}\right]\times 100\%$

    where INtest is percent infection of test compound, μINmock is average of mock, and μINvehicle is the average of infection control.

    Percent viability (PV) is calculated as follows:

    $\mathrm{P}\mathrm{V}=\frac{\mathrm{C}\mathrm{N}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}{\mu \mathrm{C}\mathrm{N}\mathrm{m}\mathrm{o}\mathrm{c}\mathrm{k}}\times 100\%$

    where CNtest and μCNmock are the viable cell number in the treatment group and average viable cell number in the mock group, respectively.

    After normalization, the 50% efficient concentration (EC₅₀) and the 50% cytotoxicity concentration (CC₅₀) are calculated with nonlinear regression using GraphPad Prism® software. Selective index (SI) is calculated by dividing CC₅₀ by EC₅₀.

Figure 8.

Compounds having antiviral activity were examined for efficacy and cytotoxicity using immunofluorescence imaging

(A) A 384-well plate can be designed for immunofluorescence imaging as outlined.

(B) In the immunofluorescence images, red signals produced by the staining with Hoechst 33342 reflect viable cells, and green signals produced from staining with antibody against viral protein reflect viral progression. Anti-spike antibody, anti-spike S1 antibody, and anti-nucleocapsid antibody are used for marking MERS-CoV, SARS-CoV, and SARS-CoV-2, respectively. This figure is reproduced from Figure 4C of Jeong et al.¹

(C) In the dose response curve, green dots represent relative infection rate, while blue dots represent relative cytotoxicity at various compound concentrations. Each data point represents the mean of duplicate assays with ±SD.

Expected outcomes

The protein purification procedure should yield protein close to the amount specified below:

Culture volume	Amount of protein	Yield (mg per L culture)
200 mL	2 mg	10 mg/L
500 mL	4 mg	8 mg/L
1.5 L	15 mg	10 mg/L

The ideal concentrations of substrate and enzyme in PLpro enzyme assay should yield fluorescence signal of close to 1 × 10⁴ RFU (relative fluorescence unit) (Figure 5B). A well-performed high-throughput screen should result in average Z′ score of higher than 0.7 (Figure 7B), and immunofluorescence assay should result in images as shown in Figure 8B.

Limitations

One limitation of our pipeline in which an in vitro protease assay is combined with an immunofluorescence-based infection assay is that these protocols are designed to specifically identify antivirals that target the viral proteases. Therefore, adaptation of this protocol to other in vitro protein assays may require additional optimization efforts. However, we believe this is addressable since the fluorescence probe AMC is applicable to various types of protease reactions generated by peptide cleavage. Moreover, the protocol for our infection assay can be easily used to monitor viral infection under given conditions of interest. With further optimization, including addition of read-outs for cell metabolism or homeostasis of mitochondria or target proteins, our current protocol for infection assay can be applicable to many purposes other than examination of viral infection.

Troubleshooting

Problem 1

Insufficient signal intensity from the AMC-peptide reaction with viral protease (related to step 4.c.xi).

Potential solution

If SARS-CoV-2 PLpro is the protease being used, then the sequence of the AMC-peptide substrate would not be an issue. Instead, insufficient signal or high background might be due to low purity of the substrate. To address this issue, the AMC-peptide should be resynthesized and purified. If viral protease other than that of SARS-CoV-2 are to be used, a panel of AMC-peptide substrates designed based on the native recognition sites of the protease of interest should be screened to select an optimal substrate for the assay.

Problem 2

Insufficient signal intensity with newly prepared AMC-peptide substrate (related to step 4.c.xi).

Potential solution

Insufficient or no signal might be due to poor activity of the recombinant protease, PLpro. To address this issue, the PLpro protein should be tested with the native viral polyprotein. In the case of SARS-CoV-2, 67 kDa viral polyprotein is known to be cleaved by the protease into 25 and 42 kDa units. Active protease should produce these cleavage products upon incubation with the viral polyprotein; otherwise, the recombinant protease protein needs to be newly prepared.

Problem 3

High background in immunofluorescence images (related to step 12.h).

Potential solution

The viral infection assay requires a primary antibody specific to the viral epitopes. Therefore, poor specificity to the viral protein (specifically spike protein in our assay) can result in high background due to non-specific promiscuous binding to the host cell proteins. To address this issue, several available antibodies should be screened for selection of antibody that produces highest specific signal in a given condition. For example, instead of using monoclonal antibody as in this protocol, a polyclonal antibody (i.e., Sino Biological Cat# 40070-T62 for MERS-CoV) can be tested for the increase in signal production. Reversely, since a polyclonal antibody has been used to tag SARS-CoV-2 nucleocapsid protein in this protocol, a monoclonal antibody (i.e., Sino Biological Cat# 40592-R505) can be tested for the increase in signal production. High background can result from inappropriate signal gain in the plate reader. In this case, signal gain or sensor probe needs to be adjusted/optimized to reduce the background signal.

Problem 4

Unclear efficacy measures (e.g., IC₅₀, CC₅₀) of test compounds in the immunofluorescence infection assay (related to step 13.z).

Potential solution

The efficacy of antiviral compounds may not be the same at different MOI. Therefore, determination of optimal MOI is critical to have an appropriate test window for each compound. Before proceeding to the actual experiment, the cytotoxicity of compounds should also be determined under the determined condition (e.g., host cell line, duration of compound treatment, etc), and the test window should be designed based on the cytotoxicity profile. Adjusting viral MOI or compound concentrations can solve the problem of unclear efficacy measures for the test compounds.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Wonsik Lee (wonsik.lee@skku.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.