Cell culture and drug treatment
The African green monkey kidney epithelial cell line Vero E6-ACE2 was cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS; Gemini Bio), 1% penicillin/streptomycin (pen/strep) (Corning Inc.) , and 1 μg/ml puromycin. The human non-small cell lung adenocarcinoma cell line H1299 was cultured in RPMI-1640 medium containing 10% FBS and 1% pen/strep. All cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. HA15 was purchased from Sigma Aldrich and dissolved in DMSO. Vero E6-ACE2 cells were treated with HA15 at indicated concentrations and 1% DMSO was used as a control. The Vero E6 and H1299 cell lines were obtained from ATCC.
The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281. The virus was propagated in Vero E6-ACE2 in a DMEM media supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco) and 0.5 µg/ml TPCK-treated trypsin (Worthington Biochemical). When 90% CPE was confirmed, supernatant was collected and passed through a 0.45 μm pore size polyethersulfone filter and aliquoted and stored at −80 °C until further use. The virus titer was determined by plaque assay.
Plaque reduction assay
Evaluating the anti-viral activities of HA15 was done by plaque reduction assay as described previously18. Confluent monolayers of Vero E6-ACE2 cells in 6-well plates were washed once with DMEM and infected with approximately 100 plaque-forming units (PFUs) of SARS-CoV-2 in each well. The plates were incubated at 33 °C for 45 min for virus adsorption. The virus inoculum was then removed and replaced by overlay media (DMEM containing 1% low-melting agarose without serum) containing 2-fold serial dilutions of HA15 and placed in 33 °C CO2 incubator for 72 hr when plaques can be visualized under light. The cells were fixed with 4% formaldehyde solution for at least 30 min and the overlaid agarose was removed. The cells were stained with 0.2% (w/v) crystal violet solution. The plaques were counted by visual examination and the size of the plaques were measured by scale loupe.
Virus infection and cell harvest
Vero E6-ACE2 and H1299 cells were seeded in 6-well plates and allowed to attach. Cells were washed once with fresh DMEM and infected with 0.5 or 3 MOI of SARS-CoV-2 in each well. DMEM media was used as a mock infection. The plates were incubated on a rocker at 33 °C for 45 min for virus adsorption. The virus inoculum was then removed and replaced by DMEM media and placed in 33 °C CO2 incubator The cell pellets were collected at indicated time points and stored at −80 °C. The cell pellet samples were lysed, and cell lysates were subjected to Western blot analysis or RT-qPCR for the detection of the interest of the proteins or RNA of RNA.
Transfection of siRNAs and plasmid DNA expression vector
For siRNA knockdown, the cells were transfected with Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) with 60pmol of siRNAs (Dharmacon-GE Healthcare) according to the manufacturer’s recommendation. The control siRNA and siRNAs targeting GRP78 have been previously described19 and their sequences are as follows: siCtrl: GAGAUCGUAUAGCAACGGUdTdT; si78#1: GGAGCGCAUUGAUACUAGAdTdT; si78#2: CUUAAGUCUCGAAUGUAAUdTdT. The construction of the expression plasmid for FLAG-GRP78 (F-GRP78) has been previously described20. The empty vector pcDNA3 or the expression vector for F-GRP78 was transfected into Vero E6-ACE2 and H1299 cells using the BioT Transfection Reagent (Bioland Scientific) following the manufacturer’s recommendation. The cells were incubated with the transfection mix for 48 hr before harvesting cells for immunoblot analysis of the protein of interest.
Preparation of cell lysates and immunoblot analysis has been described previously21. Proteins were electrophoresed in 8% or 10% SDS-PAGE gels, transferred to Nitrocellulose membrane, and probed with the following antibodies. Primary antibodies: mouse anti-GRP78 (1:1000, BD Biosciences, 610979), mouse anti-GAPDH (1:1000, Santa Cruz Biotechnology, Inc., sc-32233), mouse anti-SARS-CoV-2 Spike Protein ( S1-NTD) (1:1000, Cell Signaling, #42172). Secondary antibody: mouse IgG1 binding protein conjugated to HRP (1:1000, Santa Cruz Biotechnology, Inc., sc-525408). HRP signal was detected by SuperSignal West Pico Chemiluminescence substrate (Thermo Fisher Scientific, 34080) and protein bands were visualized by ChemiDoc XRS + imager (Bio-Rad Laboratories) and quantified by Image Lab software version 4.0.1 build 6 (Bio-Rad Laboratories) ).
RNA extraction and reverse transcription quantitative real-time PCR
Vero E6-ACE2 cells were mocked infected or infected with SARS-CoV-2 at 0.5 or 3 MOI for 12 or 24 hours. The cells were lysed with TRI reagent (Millipore-Sigma, Burlington, MA) and total RNA was extracted according to the manufacturer’s recommendations. 1 μg of total RNA was used to synthesized cDNA using the qScript cDNA Supermix (QuantaBio, Beverly, MA) following manufacturer’s protocol. Quantitative real-time PCR analysis of GRP78 and GAPDH mRNA was conducted using the KAPA SYBR FAST qPCR Master Mix (Roche Sequencing and Life Science, Wilmington, MA) and analyzed by the Stratagene MX3000P Real-Time qPCR System (Agilent, Santa Clara, CA). The PCR conditions are 40 cycles, 15 s at 95 °C, 15 s at 60 °C, 15 s at 72 °C. The sequences for the primers used in this study are as followed: GRP78 5′-GTCAGGCGATTCTGGTCATT-3′ and 5′-GGTGAAAGACCCCTGACAAA-3′, GAPDH 5′- TGCACCACCAACTGCTTAGC-3′ and 5′-GGCATGGACTGTGGTCATGAG-3′.
WST-1 cell viability assay
Vero E6-ACE2 cells were seeded at a density of 10,000 cells per wells in a 96-well plate with DMEM media containing 10% FBS and 1% pen/strep. The cells were allowed to attached overnight and next day the media was removed and replaced with DMEM media containing 1% pen/strep and no FBS. The cells were then treated with DMSO or increasing concentrations of HA15 (0.625 μM to 5 μM). Cell viability was measured at 24, 48, and 72 hr post drug treatment using the (4-[3-(4-Iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]1,3-benzene sulfonate) WST-1 cell proliferation assay kit (Takara Bio USA, Inc., San Jose, CA) according to manufacturer’s recommendation. Colorimetric measurement was detected using a Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA) at a wavelength of 450 nm and subtracted by the reference wavelength of 650 nm. Background absorbance of blank media (DMEM 1% pen/strep) was also measured and subtracted from the sample reading.
Animals and in vivo procedures
K18-hACE2 transgenic mice were purchased from Jackson Laboratory (cat. no. 034860). Female mice (about 10-week-old) were used. All animal care and experiments were performed according to the NIH guidelines for the care and use of laboratory animals. All animal studies were approved by the Institutional Animal Care and Use Committee of USC. Mice (n = 3 for each group) were intranasally infected with 103 pfu of SARS-CoV-2 virus (USA-WA1/2020) plaque isolate in 30 μl PBS. HA15 (Cat. #CS-5825) was obtained from ChemScene (Monmouth Junction, NJ, USA). HA15 was first dissolved in DMSO and diluted in PBS for intraperitoneal injection. HA15 was intraperitoneally injected daily at 35 mg/kg from the time infected to day 3 post infection. On day 3, mice were euthanized by isoflurane overdose and the lung tissues were collected, homogenized, and lysed in 500 µl of TRIzol for RNA isolation and RT-qPCR analysis.
RNA extraction and RT-qPCR analysis of mouse lung tissues
Total RNA was isolated from single-cell suspended mouse tissue in 500 μl of Trizol (Thermo Fisher Scientific, Waltham, MA). RNA was extracted following the manufacturer’s protocol for the Monarch Total RNA miniprep kit (New England Biolabs, Ipswich, MA). Complementary DNA (cDNA) was generated using 100 ng of total RNA with the iScript Reverse Transcription Supermix (Bio-Rad Laboratories) following the manufacturer’s protocol. qPCR was performed in a 96-well plate with PrimeTime® Gene Expression Master Mix (Integrated DNA Technologies (IDT), Coralville, IA) in triplicate with 1 μl of cDNA using primers for SARS-CoV-2 N1 protein and murine β-actin on the Bio-Rad CFX96 Real-Time PCR Detection system. Relative gene expression of SARS-CoV-2 N protein was calculated based on a genomic equivalent (GE) standard curve using the 2019-nCoV_N_Positive Control DNA (catalog# 10006625, IDT). The GE from the experimental samples was derived from this curve using the Maestro analysis software. Pre-designed TaqMan probes were purchased from IDT: N1 [SARS-CoV-2 (2019-nCoV) CDC RUO Primers and Probes Cat# 10006713]murine β-actin (Cat# Mm.PT.39a.22214843.g).
All pair-wise comparisons were made using the two-tailed unpaired Student’s t test in Microsoft Excel. Data are presented as the mean ± Standard Error of the mean (SEM). A pvalue of ≤0.05 is considered statistically significant. All graphs were generated with GraphPad Prism v.8.3.0 (GraphPad Software, San Diego, CA).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.