Identification of ZDHHC17 as a Potential Drug Target for Swine Acute Diarrhea Syndrome Coronavirus Infection

Abstract

The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.

Keywords: 2-bromopalmitate; CRISPR-Cas9 screen; DHHC domain; SADS-CoV.

FIG 1

Identification of genes critical for SADS-CoV replication by CRISPR library screening. (A) Genome-wide CRISPR screening strategy. Cas9-expressing HeLa cells are transduced with the genome-wide CRISPR lentivirus and selected with puromycin, followed by SADS-CoV infection. Surviving cells and the mock control are harvested and sgRNA abundance is determined using next-generation sequencing. (B) Bubble plot of data from 2 independent SADS-CoV screens. Red lines denote log₂ fold change of ±2. (C) Scatterplot comparing log₂ fold change from 2 independent SADS-CoV screens.

FIG 2

Knockout of ZDHHC17 decreases the SADS-CoV replication. (A) The target sequence in clonal cells is amplified and cloned into the pGEM-T-EASY vector. (Upper) Edited nucleotide sequences in the ZD17 gene alleles are shown according to sequencing analysis. (Lower) The clonal HeLa-ZD17^KO and HeLa cells were cultured in 6-well plates, and the expression of endogenous ZD17 was detected by Western blotting with anti-ZD17 rabbit polyclonal antibody. (B) HeLa and HeLa-ZD17^KO cells were cultured in 24-well plates and infected with SADS-CoV (MOI, 0.1). At different time points (2, 12, 24, 48, and 72 hpi), RNA was extracted from supernatants and viral genome copies were determined by RT-qPCR with primers targeting the SADS-CoV RdRp gene. (C) Cells from panel B were fixed at 12, 24, and 48 hpi, respectively, and analyzed by IFA using an anti-N protein antibody. (D) The infection rates in panel C were quantified with high content analysis. (E) At 24 h and 48 hpi, CPE was examined to compare the production of infectious progeny virus. (F) The real-time growth and adhesion kinetics of HeLa and HeLa-ZD17^KO cells were monitored using a label-free cell-based assay by the xCELLigence real-time cellular analysis (RTCA) system.

FIG 3

ZDHHC17 is involved in viral RNA synthesis. HeLa and HeLa-ZD17^KO cells were inoculated with SADS-CoV (MOI, 0.1) at 4°C for 1 h and washed with cold PBS. (A) The cells were harvested and viral RNA was extracted for determining the virion attachment at the cell surface. (B) The infected cells as described above were further cultured at 37°C for another 1 h. Cells were then harvested after pronase treatment and viral RNA was extracted for determining the virion internalization. (C) At 24 hpi, the ratio of SADS-CoV RNA copy number in the supernatants versus the cell lysates were separately determined by RT-qPCR for assembly and release assay. (D) Quantification of extracellular genomic RNA. (E) Quantification of intracellular positive- and negative-strand RNA at 0, 2, 4, 6, 8, 10, and 12 hpi. (F) Viral RNA was assessed by staining cells with anti-dsRNA antibody followed by confocal microscopy analysis. (G) The infectious virions secreted from HeLa and HeLa-ZD17^KO cells were determined by 50% tissue culture infectious dose assays in Vero cells.

FIG 4

DHHC domain of ZD17 affects SADS-CoV replication. (A) Overexpression of ZD17 and truncation mutants in HeLa and HeLa-ZD17^KO cells detected using anti-S-tag mouse monoclonal antibody and HRP-conjugated goat-anti-mouse IgG. (B) HeLa and HeLa-ZD17^KO cells were transfected as described for panel A and infected with SADS-CoV (MOI, 0.1). At 48 hpi, RNA was extracted from supernatants, and viral RNA was quantified by RT-qPCR. (C) Cells in panel B were fixed at 48 hpi and analyzed by IFA using an anti-N protein antibody. Scale bar, 100 μm.

FIG 5

Effect of 2-BP treatment on SADS-CoV infection. (A) Cytotoxicity examination of 2-BP by CCK-8 assay. HeLa cells cultured in the 96-well plates were incubated with 2-BP at the indicated concentrations. At 24 h after incubation, the inhibitor was removed, and CCK-8 reagents (10 μl) were added. After another 2 h of incubation, the optical density at 450 nm was determined. (B) Expression levels of N protein at 24 hpi after treatment with 2-BP. Cells were infected with SADS-CoV at an MOI of 0.2. After 1 h of absorption, the inoculum was removed and cells were maintained in serum-free medium with 2BP. (C) Infection rates in panel B were quantified with high-content analysis. (D) At 24 hpi, RNA was extracted from supernatants and viral RNA was quantified by RT-qPCR. (E and F) Impact of 2-BP treatment on SADS-CoV infection in SIEC and ST cells.