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Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication

BMC Genomics volume 25, Article number: 796 (2024) Cite this article

Abstract

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus–host interactions and potential antiviral strategies.

Importance

FMDV is a pathogen that causes highly contagious animal disease worldwide. The WD40 domain of ATG16L1 is necessary for non-canonical autophagy, while dispensable for canonical autophagy. Thus, the WD40 knockout PK15 cell line established by present study could use as a tool to research the impact of non-canonical autophagy on FMDV replication. The results showed that knockout of the WD40 domain of ATG16L1 in PK15 cells could enhance FMDV replication. The results also showed that differentially expressed innate immune factors, such as Mx1, IFIT1, RSAD2, and IRF9, were significantly enriched in the interferon signaling pathway. Our research also indicates non-canonical autophagy inhibits FMDV replication by innate immune, which helps to elucidate the role of the non-canonical autophagy in the host antiviral strategies.

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Introduction

Foot-and-mouth disease (FMD) is an acute, highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV), which mainly infects cloven-hoofed livestock and wild animals [1, 2]. This disease is characterised by vesicular lesions in the buccal cavity, feet, and teats and can cause severe losses to agricultural development. FMDV is a member of the Aphthovirus genus within the family Picornaviridae, its genome encodes 12 proteins, including four structural proteins (VP1 to -4) and eight nonstructural proteins (Lpro, 2 A, 2B, 2 C, 3 A, 3B, 3 C, and 3D) [3]. FMDV has seven serotypes, named O, A, C, Asia 1, SAT1, SAT2 and SAT3. In addition, multiple subtypes have further evolved from each serotype [4]. FMD vaccine development faces many challenges because there is no cross-protection between different serotypes [5]. It has been reported that FMDV infection induced macroautophagy/autophagy, which was beneficial for virus replication. FMDV VP1 degrades host proteins YTHDF2 through an AKT-MTOR-dependent autophagy to regulate IRF3 activity for viral replication [6]. FMDV VP2 interacted with HSPB1 and activated the EIF2S1-ATF4 pathway, resulting in autophagy and enhanced FMDV replication [7]. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy to promote viral replication [8]. FMDV VP3 also interacted with HDAC8 and promoted its autophagic degradation to facilitate viral replication [9]. FMDV 3 A protein caused upregulation of autophagy-related protein LRRC25 to inhibit IFN signaling and counteract innate antiviral responses by autophagy [10].

LC3-associated phagocytosis (LAP) is a non-canonical autophagy through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events. In vivo, ATG16L1 is a critical mediator of canonical autophagy that plays an important role in the formation of the LC3 lipidation complex and is responsible for the correct targeting of LC3 to autophagosome membranes [11, 12]. It contains an N-terminal domain that associates with ATG5 and ATG12, a large C-terminal domain (CTD) formed by 7 WD40-type repeats, and a coiled-coil domain (CCD) that binds FIP200 and WIPI2b [13, 14]. Deletion of FIP200 binding domain prevents ATG16L1 recruitment to forming autophagosomes [15, 16]. The name WD40 comes from the conservative WD dipeptide and the length of approximately 40 amino acid residues in a single repeat [17]. WD40 domain-containing proteins are abundant in eukaryotic organisms, excluding yeast ATG16, and are rarely present in prokaryotes, although a few cases have been reported in bacteria [18, 19]. Although the function of the WD repeat-containing C-terminal domain (WD40 CTD) has remained unclear, the WD40 repeat sequence is not associated with the canonical autophagy pathway because the ATG16L1 version lacking this region is fully capable of maintaining canonical autophagy in mammalian cells [14, 20]. Moreover, recent studies have revealed that the WD40 CTD is critical for non-canonical autophagy activities; that is, the WD40 CTD distinguishes between canonical autophagy and non-canonical autophagy machinery. Research has shown that the WD40 CTD of ATG16L1 is necessary for LC3 recruitment to endolysosomal membranes during non-canonical autophagy but dispensable for canonical autophagy [21]. Studies have shown that the activation of non-canonical autophagy is dependent on the WD40 CTD during influenza A virus (IVA) infection and the reduction in MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD. IAV is controlled within epithelial barriers, where non-canonical autophagy reduces IAV fusion with endosomes and the activation of interferon signaling [21]. The structure of the WD40 CTD has recently been solved; although there is some evidence that the WD40 CTD can bind ubiquitin and other factors involved in lysophagy and some forms of xenophagy, its biological function remains unclear [14, 22, 23]. WD40 mediates ATG16L1-promoted lysosomal degradation of TNFAIP3, activates the NF-κB response, and inhibits autophagy, which has important implications for novel roles in protein stability and inflammatory signaling [24].

PK15 cells were isolated from the kidneys of adult pigs by Stice E. et al. in 1955; these cells are clones of PK1a cells that can be used for the proliferation and characterisation of various viruses. The most common application of PK15 cells is porcine circovirus type 2 (PCV2) culture and inactivation for vaccine preparation; these methods are also widely used in the study of porcine parvovirus (PPV) [25, 26], porcine pseudorabies virus (PRV) [27], senecavirus A (SVA) [28] and FMDV [29] .

In this study, a WD40 knockout (KO) PK15 cell line was established by using CRISPR/Cas9 technology and evaluated via a series of methods. Then, bioinformatics analysis of the host‒virus interaction of FMDV was performed using transcriptome RNA‒seq. Finally, the genes differentially expressed between WT-PK15 and WD40-KO-PK15 cells were selected for qRT‒PCR to validate the RNA‒seq data. The results showed that WD40 plays an important role in the innate immune response, and the WD40-KO cell line can also be used in research on foot-and-mouth disease and vaccine production.

Results

Successfully established WD40 knockout PK15 cell line

To investigate the effect of WD40 knockout on FMDV replication, WD40 knockout PK15 cells (WD40-KO-PK15) were first established using the CRISPR/Cas9 system. The gRNA expression plasmid was constructed and packaged to generate lentiviral particles to infect cells. After screening the cells, puromycin-treated cells were selected, and the genomic DNA was extracted for sequencing. One base was knocked out in the knockout cell line compared with the WT-PK15 cell line (Fig. 1A‒B). The expression of WD40 in the cell line was confirmed by Western blotting. The results showed that WD40 was successfully knocked out in the WD40-KO-PK15 cell line compared with the WT-PK15 cell line (Fig. 1C). To test whether knockout of the WD40 gene has an effect on the normal growth of host cells, a cell growth curve was generated. The results showed that the growth rate of WD40-KO-PK15 cells was the same as that of WT-PK15 cells (Fig. 1D), indicating that knockout of the WD40 gene did not affect the normal growth of host cells. In conclusion, these results indicate that the knockout cell line was successfully established.

Fig. 1
figure 1

Identification of WD40 knockout PK15 cells. (A) Alignment of the WT-PK15 and WD40-KO-PK15 sequences using DNAMAN software. The red box indicates the sgRNA sequence and the mutations in PAM motif. (B) Snapgene software was used to confirm the sequencing peaks of PCR products WT-PK15 and WD40-KO-PK15. (C) The expression of ATG16L1 was detected by Western blotting with ATG16L1 antibody, and β-actin was used as a control to show the even loading of samples. (D) WT-PK15 and WD40-KO-PK15 cells were seeded in 24-well plates at a density of 2.8 × 105 cells/well. From 12 to 72 h after seeding, the cells were trypsinized and counted every 12 h. The experiment was repeated three times with similar results. Data are expressed as mean ± SD (n = 3)

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We next examined whether knocking out WD40 expression affected the growth and morphology of the cells. After stable passage for 3 generations, the growth and morphology of the two cell lines were observed via optical microscopy. Karyotype tests were used to determine changes in the structure and number of chromosomes in WD40-KO-PK15 and WT-PK15 cells. As shown in Fig. 2A, there was no significant difference in cell morphology; cells had irregular polygonal shapes with clear edges, uniform sizes and good growth conditions. Karyotype analysis also revealed no significant differences in chromosome structure or number between WD40-KO-PK15 and WT-PK15 cells (Fig. 2B‒C). Therefore, deletion of WD40 does not cause morphological changes or chromosomal aberrations in cells.

Fig. 2
figure 2

Cell morphological analysis. (A) After stable passage for 3 generations, the growth morphology of WT-PK15 and WD40-KO-PK15 cells was observed by optical microscope with three days. Chromosomal analysis of WT-PK15 (B) and WD40-KO-PK15 (C) cell lines

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WD40 knockout enhances FMDV Replication

The cytopathic effect (CPE) refers to the activity and morphological changes in host cells caused by viral infection. Both WD4O-KO-PK15 cells and WT-PK15 cells were infected with FMDV (MOI = 0.1) for 8 h, after which the CPE was observed under an optical microscope. Compared with WT-PK15 cells, WD4O-KO-PK15 cells had greater CPE (Fig. 3A). FMDV-positive WD4O-KO-PK15 cells were also observed by using immunostaining and confocal microscopy, as shown in Fig. 3B. To verify the results, the number of VP1-positive cells was quantified via fluorescence microscopy, and the relative value of the average fluorescence intensity in the field of view was calculated. As shown in Fig. 3C, the relative value of the average visual field fluorescence intensity of WD40-KO-PK15 cells was approximately 2.5 times that of WT-PK15 cells.

Fig. 3
figure 3

Growth characteristics of FMDV. (A) CPE caused by FMDV, WT-PK15 and WD40-KO-PK15 cells was seeded in 6-well plates (2 × 106 cells/well) and were infected with FMDV at an MOI of 0.1 for 8 h. (B) WT-PK15 and WD40-KO-PK15 cells infected with FMDV (MOI = 1.0) or without FMDV for 2 h. The signal of FMDV protein VP1 was observed under confocal microscopy by immunofluorescence assay. Red signals represent VP1 and nucleus was stained with DAPI. (C) The number of VP1 positive cells was quantified by fluorescence microscopy. The relative value of the average fluorescence intensity of visual field was calculated. The figure shows the data in 3 fields, and the bar chart represents the average relative value of the mean field fluorescence intensity. Statistical significance was analyzed by Student’s t-test: ***P < 0.001

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To investigate the replication of FMDV in these two cell types, WD40-KO-PK15 cells and WT-PK15 cells were infected with FMDV. Figure 4A shows that the expression of the FMDV VP1 protein and LC3 II was increased in WD40-KO-PK15 cells. The viral mRNA levels of FMDV were tested by qRT‒PCR at different times and at different doses (Fig. 4B‒C). Viral titres were determined and compared between WD40-KO-PK15 cells and WT-PK15 cells (Fig. 4D). These results showed that the FMDV replication level in WD40-KO-PK15 cells was significantly greater than that in WT-PK15 cells, which is also consistent with the growth characteristics of FMDV.

Fig. 4
figure 4

WD40 knockout enhances FMDV replication. (A) WT-PK15 and WD40-KO-PK15 cells were infected with equal amounts of FMDV (MOI = 1.0) for 0.5, 1, 2, and 4 h. The expression of FMDV VP1 protein, ATG16L1, LC3 was determined by Western blotting, and β-Actin was used as an internal reference. (B) The cells of WT-PK15 and WD40-KO-PK15 were infected with equal amounts of FMDV (MOI = 1.0) for 1, 4 and 8 h. qRT-PCR was performed to examine the expression of FMDV 3D relative mRNA. (C) WT-PK15 and WD40-KO-PK15 cells were infected with different amounts of FMDV at MOI of 0.2, 1.0, and 5.0 for 8 h. qRT-PCR was performed to examine the expression of FMDV 3D relative mRNA. (D) FMDV titers were determined by the TCID50 method after cells were infected with FMDV (MOI = 0.1). All experiments were repeated three times, with similar results. Data are presented as mean ± SD (n = 3). Statistical significance was analyzed by Student’s t-test: **p < 0.01

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To confirm the above results, RNA interference was performed in PK15 cells. The knockdown effects were confirmed by Western blotting analysis (Fig. 5A). As shown in Fig. 5A‒B, compared with those in the control siRNA-transfected cells (NC), the viral protein and FMDV mRNA levels were significantly increased. In brief, the above results indicated that knockdown of WD40 significantly enhanced FMDV replication in PK15 cells.

Fig. 5
figure 5

The knockdown of WD40 by siRNA significantly enhanced FMDV replication in PK-15 cells. PK-15 cells were transfected with control siRNA (NC) or siRNA targeting to WD40 for 24 h, and then infected with FMDV (MOI = 1.0) for 6 h. (A) Western blotting analysis was performed to detect the expression of ATG16L1 and viral proteins VP1 with indicated antibodies, and β-Actin was used as an internal reference. (B) Total RNA was extracted and qRT-PCR was performed to detect the expression of FMDV 3D relative mRNA. All experiments were repeated three times, with similar results. Data are presented as mean ± SD (n = 3). Statistical significance was analyzed by Student’s t-test: *p < 0.05, **p < 0.01

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Differences in gene expression between WT-PK15 and WD40-KO-PK15 with FMDV infection

The DEGs involved in multiple reactions in WT-PK15 and WD40-KO-PK15 cells infected with FMDV were collected and analysed to explore whether the use of WD40-KO-PK15 cells is an effective tool for FMDV research. The samples underwent strict quality control and filtering to ensure subsequent accurate analysis, and finally, clean data were obtained. A total of 3982 DEGs were detected. Compared with those in WT-PK15 cells, 1809 DEGs were upregulated, and 2173 DEGs were downregulated in WD40-KO-PK15 cells; these genes were involved in 23 biological processes and 50 signaling pathways (Fig. 6A‒B). The results of KEGG analysis of signaling pathways are shown in Fig. 6B, with 51 DEGs related to autophagy-animal and 19 DEGs related to other autophagy. There were 24 DEGs involved in NOD-like receptor signaling pathway, 21 DEGs involved in PI3K-Akt signaling pathway, 19 DEGs involved in RIG-I-like receptor signaling pathway, 18 DEGs involved in Toll-like receptor signaling pathway, 17 DEGs involved in mTOR signaling pathway, 14 DEGs involved in TNF signaling pathway, 10 DEGs involved in endocytosis and 4 DEGs related to lysosome. In addition, MAPK signaling pathway, NOD-like receptor signaling pathway and NF-κB signaling pathway, etc. were also involved. The heatmaps of DEGs in WT-PK15 and WD40-KO-PK15 cells are shown in Fig. 6C. Differential gene expression was analysed by qRT‒PCR to verify the transcriptome analysis results. The immune response-related genes CXCL1, CXCL8, IRF9, Mx1, CCL5, IFIT1, IFITM3, BST2, TNFRSF17, OASL, GBP1 and RSAD2 were randomly selected for analysis and comparison in FMDV infection in WT-PK15 and WD40-KO-PK15 cells (Fig. 7), and the results were consistent with the transcriptome analysis.

Fig. 6
figure 6

Difference of gene expression between WT-PK15 and WD40-KO-PK15 with FMDV-infected. WT-PK15 and WD40-KO-PK15 cells were infected with FMDV (MOI = 0.1). The transcriptome RNA-seq and scatterplots of significant enrichment items of differentially expressed genes cluster map (A) and KEGG analyses (B) associated with were compared between WT-PK15 and WD40-KO-PK15 cells. (C) Heat maps of differentially expressed genes in WT-PK15 and WD40-KO-PK15 cell

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Fig. 7
figure 7

Verification of RNA-seq data with RT-qPCR. Validation of the differentially expressed genes using the qRT-PCR assay in WT-PK15 and WD40-KO-PK15 cells were infected by FMDV (MOI = 0.1) for 6 h. All experiments were repeated three times, with similar results. Data are presented as mean ± SD (n = 3). Statistical significance was analyzed by Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001

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Discussion

In recent decades, a series of cell lines, including hamster kidney cell line (BHK-21), and swine kidney cell line (PK-15, IBRS-2 and SK-6) were established as most widely used cell model systems for FMDV separation, culture and research [6, 8, 9, 30, 31]. In addition, porcine origin cell line (LFBK) and fetal goat tongue cell line (ZZ-R 127) also have been shown to be highly sensitive to FMDV [32, 33]. Further, Mao et al. established a stable bovine thyroid cell line (hTERT-BTY) from primary bovine thyroid (BTY) cells by telomerase reverse transcriptase overexpression, which could be used for FMDV separation, culture and detection [34]. Hou et al. established a HDAC9 knockout (KO) BHK-21 cells using CRISPR/Cas9 technology which could enhance FMDV replication [35]. In this study, a WD40-KO-PK15 cell line was constructed using the CRISPR/Cas9 system, and the shape, size and cell viability of the cells were compared with those of wild-type cells, including chromosome number and karyotype analysis. The results showed that WD40-KO-PK15 cells and WT-PK15 cells had no significant differences in morphology or growth characteristics, and knockout of the WD40 domain of ATG16L1 enhances FMDV replication.

LAP is a non-canonical function of the autophagy machinery, in which LC3 is conjugated to rab5-positive phagocytosis, using a portion of the canonical autophagy pathway. More and more evidence suggests that ATG16L1 plays an important role in both autophagy, non-canonical autophagy and innate immunity [36, 37]. The WD40 CTD of ATG16L1 is necessary for LC3 recruitment to the inner lysosomal membrane for non-canonical autophagy, but it is not necessary for canonical autophagy [13, 38]. ATG16L1 is observed in large homodimer complexes consisting of ATG12-ATG5-ATG16L1 dimerized by the CCD domain in higher eukaryotes. ATG12-ATG5-ATG16L1, ATG7 and ATG3 mediate LC3 conjugation to phosphatidylethanolamine (PE) recruitment to double-membrane phagophores in canonical autophagy, while ATG12-ATG5-ATG16L1, ATG7 and ATG3 conjugate LC3 to both PE and phosphatidylserine (PS) in LAP [39, 40]. ATG16L1 middle region and residues within the CCD can interact with inducers of autophagy FIP200 (the ULK1 adaptor protein), WIPI2 (PI3P-binding molecule), PI3P, or Rab33b which is benefit for autophagosome formation and maturation in canonical autophagy [15, 16, 41, 42]. ATG16L1 WD domain can interact with inducers of LAP Rip2 (an adaptor protein of intracellular pattern recognition molecules Nod1 and Nod2), TMEM59 (a transmembrane protein), and vATPase (vacuolar adenosine triphosphatase, a multi-subunit proton pump that plays a crucial role in organelle acidification) in innate immunity which damage cytokine response in LAP during bacterial infection, and disrupt the interactions with the ATG16L1 WD domain facilitates bacterial and viral immune evasion and virulence [14, 43,44,45,46,47].

LAP can effectively phagocytose and eliminate invasive pathogens via the participation of the BECN1 complex [48], and NOX2, P40PHOS, and rubicon are exclusive to LAP. In autoimmune diseases, LAP contribute to MHC class II presentation of autoantigens, thereby amplifications of the autoreactive CD4 T cell response [49]. Bone marrow mesenchymal stem cells limit bacterial pneumonia infection by enhancing the LAP of macrophages [50]. Mice with systemic loss of non-autophagy machinery are highly susceptible to low-pathogenicity IAV, which causes extensive viral replication throughout the lung and cytokine amplification, leading to fulminant pneumonia, lung inflammation and high mortality [21]. SVA infection in PK15 cells induces glycolysis, which modulates the regulatory effect of glycolysis on the replication of SVA mainly via RIG-I signaling, as indicated by the significantly increased expression of hexokinase 2 (HK2), 6-phosphofructokinase (PFKM), pyruvate kinase M (PKM), phosphoglycerate kinase 1 (PGK1), hypoxia-inducible factor-1 alpha (HIF-1α), and superoxide dismutase-2 (SOD2) [51]. Studies have shown that FMDV-infected PK15 cells promote the transfer of mTOR to lysosomes to enhance the interaction between mTOR and Rheb and activate the PI3K/AKT/TSC2/Rheb/mTOR/p70S6K1 pathway to promote viral replication [52], which is consistent with the results of RNA‒seq in this study.

Interleukin, interferon, the tumour necrosis factor superfamily and other cytokines act in concert with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response [53]. The interferon-regulated Mx1 gene encodes myxovirus resistance protein A (MxA), a large kinetic protein-like guanosine triphosphatase (GTPase), an antiviral limiting factor that is active against a variety of RNA and DNA viruses, including IAVs [54]. Type I interferons have powerful antiviral functions and can induce the expression of many antiviral-related proteins by activating the JAK/STAT pathway, thus inhibiting the virus. When the body is infected by pathogenic microorganisms such as viruses, IRF9 is activated to participate in the JAK/STAT pathway, type I IFN interferon signaling, and control viral infection [55, 56]. In KEGG analysis, IRF9 was significantly enriched in NOD-like receptor signaling pathway, CXCL8 was significantly enriched in RIG-I-like receptor signaling pathway, cytokine-cytokine receptor interaction and Toll-like receptor signaling pathway following infection with FMDV. At the same time, GBP1 was significantly enriched in autophagy and CCL5 was significantly enriched in TNF signaling pathway during FMDV infection. Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that can be induced by viral infection and interferons (IFNs). This factor can inhibit the entry and replication of many viruses that do not rely on receptor usage but rely on processes that occur within the nuclear body [57], such as IAV [58] and vesicular stomatitis virus [59]. IFITM3 plays an important role in preventing endocytosed viral particles from accessing the host cytoplasm, inhibiting the RNA-virus-triggered production of type I IFNs and cellular antiviral responses [57]. In addition, antiviral innate immunity is the first line of defence of host cells against viral infection. In the present study, antiviral proteins such as OASL, BST2, and ISG15 showed downregulated expression in WD40-KO-PK15 cells compared with WT-PK15 cells, which was consistent with the findings of previous reports [60,61,62], These results indicate that WD40 can limit FMDV infection in PK15 through the antiviral innate immune response.

Materials and methods

Cell and viruses

Porcine kidney cells (PK15 cells) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified incubator at 37℃ with 5% CO2. The FMDV/O/HN/CHA/2012 strain was stored in the National FMD Reference Laboratory (Lanzhou, Gansu, P. R. China). FMDV was propagated in BHK-21 cells, and the virus titer was determined as the 50% tissue culture infectious dose (TCID50), which was calculated by the Reed–Muench formula [63]. All virus-related experiments were conducted in the Biosafety Level-3 (BSL-3) Laboratory of Lanzhou Veterinary Research Institute following the standard protocols and biosafety regulations provided by the Institutional Biosafety Committee.

Establishment of the WD40 knockout PK15 cell line

WD40 knockout PK15 cells (WD40-KO-PK15) were established using the CRISPR/Cas9 system. To construct the gRNA expression plasmid, complementary oligonucleotides encoding gRNAs were annealed and cloned and inserted into the BsmB I (Fermentas) site in lentiCRISPRv2 (Addgene). Then, lentiviral particles were generated by transfecting HEK293T cells with the lentiCRISPRv2-gRNA construct psPAX2 and pMD2. Cells were seeded at ~ 40% confluency and transduced with lentivirus via spinfection in 12-well plates. Twenty-four h after infection, the cells were detached with trypsin (Beyotime) and incubated at a low density; the selection agent was added 3 h after plating. The cells were treated with the indicated concentrations of puromycin (Beyotime). The media were changed the next day, and the cells were passaged every other day. Four days later, the cells were harvested for extraction of genomic DNA for sequencing. The primers used for plasmid construction and sequencing are listed in Table 1.

Table 1 The primers of WD40 used for plasmid construction and sequencing
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Western blotting

Cells were lysed using phenylmethylsulfonyl fluoride (PMSF Sigma) lysis buffer and clarified by centrifugation. Protein samples were separated via precast 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gel, transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% skim milk in TBST for 1 h. The membranes were incubated overnight at 4℃ with antibodies against ATG16L1 and LC3A/B (Cell Signaling Technology 8089, 41085), VP1 (generated in our laboratory [64]) and β-actin (Thermo MA5-32540). Primary antibodies were detected using HRP-conjugated secondary antibodies (Thermo 31430, 31460) for 1 h. The protein bands were detected with enhanced chemiluminescence detection reagents (Thermo) and visualised with Image Lab 4.1 (Bio-Rad).

Viral infection, RNA extraction, and qRT‒PCR

PK15 cells were cultured in a 60-mm dish to reach approximately 90% confluence. Afterwards, the cells were washed with PBS and incubated with FMDV at a multiplicity of infection (MOI = 1.0) at 37℃ for 0.5 h, washed with PBS, and cultured in 2 mL of 1% FBS DMEM. After infection for 6 h, the supernatant was removed, the cells were washed again with PBS, and 1 mL of TRIzol Reagent (Invitrogen) was subsequently added to each dish. Total RNA was isolated according to the manufacturer’s instructions. RNA (1 µg) was used as the template for cDNA synthesis via the use of HiScript II Q RT SuperMix for qPCR (Vazyme). cDNA was then subjected to real-time PCR quantification using Taq Pro Universal SYBR qPCR Master Mix (Vazyme). The GAPDH gene was used as an internal control. The primers used in the experiment are listed in Table 2. All the experiments were repeated at least three times, and relative mRNA expression levels were calculated using the threshold cycle (2−△△Ct) method.

Growth characteristics of Foot-and-Mouth Disease Virus in WD40-knockout cells

The distribution of FMDV in WD40-KO-PK15 cells was detected via indirect immunofluorescence assay (IFA). WT-PK15 and WD40-KO-PK15 cells were infected with FMDV (MOI = 1.0) for 2 h. The cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 3% BSA at 37℃ for 30 min. The cells were incubated with an anti-VP1 antibody (1:1000 dilution in 1% BSA) at 4℃ overnight. Then, the cells were incubated with a goat anti-rabbit IgG 594 antibody (1:500 dilution in 1% BSA) at room temperature for 2 h, after which the nuclei were stained with DAPI. Finally, the cells were observed by confocal microscopy.

Table 2 Primers used for qRT‒PCR amplification
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TCID50 assay

Viral titer was determined using a TCID50 assay. PK15 cells were seeded in 96-well plates at 90% confluence, and a series of 10-fold serial dilutions from 10− 1 to 10− 8 of the virus samples were prepared in another plate. One hundred microliters of the above samples was added to each well, and the plates were incubated at 37℃ for 1 h. Then, the inoculum was removed, and the cells were cultured in DMEM supplemented with 1% FBS for 72 h. The TCID50 was calculated by the Reed–Muench method.

Transcriptome RNA‒seq

The differential expression of mRNAs in FMDV-infected and uninfected wild-type (WT-PK15) and WD40-KO-PK15 cells was detected via RNA sequencing (RNA‒seq) to identify the pathways associated with innate immunity. The cells were infected with FMDV at an MOI of 0.1 for 6 h. The samples were washed three times with PBS, and total RNA was extracted using TRIzol reagent. These samples were analysed at the transcriptome level using RNA‒seq at BIOTREE (ShangHai, China).

Karyotype Analysis

WT-PK15 and WD40-KO-PK15 cells were treated with 0.1 µg/mL colchicine for 6 h at 37℃ to obtain intermediate cells, after which the chromosome number was determined.

Conclusion

In summary, our study showed that WD40, an indispensable component of non-canonical autophagy, could regulate the replication of FMDV at PK15 and play an important role in host antiviral immunity. Further study of the relationship between WD40 and FMDV could provide support for the prevention and control of FMD and aid in the production of FMD vaccines.

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA015011) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/.

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Acknowledgements

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Funding

This work was supported by the National Key Research and Development Program of China (grant no. 2021YFD1800300), the Key Technologies R&D Program of Gansu Province (grant no. 21ZD3NA001-5 and 22ZD6NA001), the Natural Sciences Foundation of Gansu Province (grant no. 23JRRA549 and 22JR5RA030), the Project of National Center of Technology Innovation for Pigs (NCTIP-XD/C03), the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2022SDZG02), Gansu Province Top Leading Talent Training Project, 2023 Basic Research on Innovation of Chinese Academy of Agricultural Science, and the earmarked fund for CARS-35.

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Authors and Affiliations

  1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, China

    Xiuping Wu & Wenhui Wang

  2. State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou Veterinary Research Institute, Lanzhou University, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China

    Xiuping Wu, Yang Yang, Yi Ru, Rongzeng Hao, Dongmei Zhao, Ruifang Ren, Bingzhou Lu, Yajun Li, Shengzhen Sun & Haixue Zheng

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  1. Xiuping Wu
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Contributions

X.W. completed the experiment, data analysis and manuscript writing. X.W. and Y.Y. design experiments and assist in preparing and revising manuscripts. Y.R., R.H., D.Z., R.R., B.Z., Y.L. and S.S. provided assistance for the experimental work, H.Z. and W.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Haixue Zheng or Wenhui Wang.

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Wu, X., Yang, Y., Ru, Y. et al. Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication. BMC Genomics 25, 796 (2024). https://doi.org/10.1186/s12864-024-10703-6

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BMC Genomics

ISSN: 1471-2164

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