viruses Article
HDAC-Specific Inhibitors Induce the Release of Porcine Epidemic Diarrhea Virus via the COPII-Coated Vesicles Ying Yang 1,2 , Huan Chen 1,2 , Caisheng Zhang 1,2 , Hyun-Jin Shin 3 , Yingjuan Qian 1,2,4, * and Yong-Sam Jung 1,2, * 1
2 3 4 *
Citation: Yang, Y.; Chen, H.; Zhang, C.; Shin, H.-J.; Qian, Y.; Jung, Y.-S. HDAC-Specific Inhibitors Induce the Release of Porcine Epidemic Diarrhea
One Health Laboratory, Jiangsu Foreign Expert Workstation, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China; 2018207012@njau.edu.cn (Y.Y.); chenhuan2019@njau.edu.cn (H.C.); 2020107034@stu.njau.edu.cn (C.Z.) MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea; shin0089@cnu.ac.kr Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Veterinary Bio-Pharmaceutical, Taizhou 225300, China Correspondence: yqian@njau.edu.cn (Y.Q.); ysjung@njau.edu.cn (Y.-S.J.); Tel.: +86-25-8439-9102 (Y.Q. & Y.-S.J.)
Abstract: Porcine epidemic diarrhea virus (PEDV) is an alpha-coronavirus causing acute diarrhea and high mortality in neo-natal suckling piglets, resulting in huge economic losses for the global swine industry. The replication, assembly and cell egression of PEDV, an enveloped RNA virus, are mediated via altered intracellular trafficking. The underlying mechanisms of PEDV secretion are poorly understood. In this study, we found that the histone deacetylase (HDAC)-specific inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), facilitate the secretion of infectious PEDV particles without interfering with its assembly. We found that PEDV N protein and its replicative intermediate dsRNA colocalize with coat protein complex II (COPII)-coated vesicles. We also showed that the colocalization of PEDV and COPII is enhanced by the HDAC-specific inhibitors. In addition, ultrastructural analysis revealed that the HDAC-specific inhibitors promote COPII-coated vesicles carrying PEDV virions and the secretion of COPII-coated vesicles. Consistently, HDAC-specific inhibitors-induced PEDV particle secretion was abolished by Sec24B knockdown, implying that the HDAC-specific inhibitors-mediated COPII-coated vesicles are required for PEDV secretion. Taken together, our findings provide initial evidence suggesting that PEDV virions can assemble in the endoplasmic reticulum (ER) and bud off from the ER in the COPII-coated vesicles. HDAC-specific inhibitors promote PEDV release by hijacking the COPII-coated vesicles.
Virus via the COPII-Coated Vesicles. Viruses 2023, 15, 1874. https:// doi.org/10.3390/v15091874
Keywords: HDAC-specific inhibitors; COPII-coated vesicles; porcine epidemic diarrhea virus; viral release Academic Editor: HuaJi Qiu Received: 1 July 2023 Revised: 31 August 2023 1. Introduction Accepted: 1 September 2023
Coronaviruses (CoVs), belonging to the order Nidovirales under the family Coronaviridae, are enveloped viruses with a positive-sense single-stranded RNA genome. The Coronaviridae family includes two subfamilies: Coronavirinae and Torovirinae. The Coronavirinae subfamily is further classified into four genera, the alpha-, beta-, gamma- and deltacoronaviruses, based on genotypic and serological characterizations [1–3]. The notorious severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent beta-coronaviruses [4]. Porcine epidemic diarrhea virus (PEDV), an alpha-coronavirus in the family Coronaviridae, causes severe watery diarrhea, vomiting, dehydration, and high mortality in neonatal suckling piglets, resulting in huge economic losses in the global swine industry [5–7]. CoVs and others enveloped viruses, use and modify intracellular compartments of the secretory pathway to facilitate their replication, assembly and egression by hijacking the
Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
Viruses 2023, 15, 1874. https://doi.org/10.3390/v15091874 https://www.mdpi.com/journal/viruses Viruses 2023, 15, 1874 2 of 16
host cell´s transport machinery [8]. Viral particles assemble in the ER-Golgi intermediate compartment (ERGIC)/Golgi compartment, and is a general hallmark of CoVs [8,9]. Previous reports revealed that alpha-coronaviruses (TGEV, HCoV-NL36) and beta-coronaviruses (MHV) assemble in the ER during the late stages of virus infection. Other studies have indicated that PEDV particles assemble in both the ER and Golgi apparatus [8,10–13]. The egress pathway of PEDV is triggered by the interaction between the nucleocapsid (N) protein loaded with the newly synthesized genomic RNA and the structural spike (S), envelope (E), and membrane (M) proteins, budding into the lumen of the ER and the ERGIC. Virus particles reach the Golgi and trans-Golgi network (TGN) by vesicular transport for glycosylation and other post-translational modifications. Mature virions are subsequently released via fusion of smooth-walled vesicles with the plasma membrane similar to exocytosis [13,14]. However, the underlying mechanisms of PEDV secretion have yet to be fully understood. In eukaryotic cells, the conventional secretory transport pathway is characterized by the sequential transport of newly synthesized lipids and proteins from the ER to the plasma membrane by transport vesicles via the ERGIC and the Golgi apparatus [15,16]. Intracellular trafficking vesicles are mainly composed of COPI-, COPII-, and clathrincoated vesicles (CCVs) [17,18]. COPII-coated vesicles mediate the transport of cargo from the ER to the Golgi apparatus [19]. COPI-coated vesicles mediate cargo transport from the Golgi apparatus to the ER, or between Golgi cisternae [20,21]. CCVs mediate cargo transport from the TGN to the endosomes and the endocytosis of cargo at the plasma membrane [17,22]. The COPII-coated machinery is composed of five cytosolic proteins: Sar1, Sec23, Sec24, Sec13 and Sec31 [23]. The assembly of COPII-coated vesicle is initiated by the recruitment and activation of the small cytoplasmic GTPase Sar1 by the GTP exchange factor (GEF) Sec12. Sar1-GTP inserts into the ER membrane and recruits the Sec23/Sec24 heterodimer by binding to the Sec23. Sec23 acts as a GTPase activating protein (GAP) for Sar1 and Sec24 participates in cargo selection. Sar1 carrying a cargo-loaded Sec23/Sec24 heterodimer forms a so-called “pre-budding complex” and in turn recruits the Sec13/Sec31 heterotetramer onto the “pre-budding complex” to complete vesicle formation [16,23–27]. As the canonical secretory pathway for ER export, COPII-coated vesicles are frequently hijacked for viral genome replication and transport of viral particles. For example, poliovirus uses COPII-coated vesicles for the formation of replication complexes (RCs) [28,29]. Parvovirus particles [30], hepatitis B subviral envelope particles [31], hepatitis C virus (HCV) lipoviroparticles [32], rotavirus NSP4 [33], and Ebola and Marburg virus matrix protein VP40 utilize the COPII transport system for intracellular transport [34]. HDAC inhibitors are approved by the United States Food and Drug Administration (FDA) as cancer therapeutics and are candidate therapies for other diseases, including arthritis, cardiac disease, inflammatory diseases, and a few neurological disorders [35–37]. In addition, HDAC inhibitors have been found to exert antiviral effects. TSA and Suberoylanilide hydroxamic acid (SAHA) suppress respiratory syncytial virus (RSV) and HCV replication [38–40]. Romidepsin prevents the entry of SARS-CoV-2 [41]. In addition, class I-selective HDAC inhibitors enhance HIV latency reversal [42]. This study investigated the relationship between HDAC-specific inhibitors and COPIIcoated vesicles during PEDV infection. We showed that the HDAC-specific inhibitors, TSA and NaB, facilitate the secretion of infectious PEDV particles. We also found that PEDV particles utilize the COPII-coated vesicles for intracellular transport. In addition, transmission electron microscopy (TEM) and immunoelectron microscopy (IEM) revealed that secretion of COPII-coated vesicles carrying PEDV virions is promoted by HDACspecific inhibitors treatment. Finally, we also demonstrated that the knockdown of Sec23A or Sec24B suppresses HDAC-specific inhibitors-induced PEDV secretion. Taken together, this study establishes that HDAC-specific inhibitors-induced COPII-coated vesicles are essential for PEDV release.
2. Materials and Methods 2.1. Cell Culture African green monkey Cercopithecus aethiops kidney epithelial cells (Vero-E6 cells) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 8% fetal bovine serum (Pan-Biotech, Inc. Aidenbach, Germany) and 1% penicillin and streptomycin (MDBio, Qingdao, China) at 37 ◦ C in a 5% CO2 incubator. 2.2. Antibodies and Reagents Rabbit polyclonal anti-Sec21p was supplied by Agrisera (Vännäs, Sweden). Rabbit polyclonal anti-COPII was purchased from Invitrogen (Carlsbad, CA, USA). Rabbit polyclonal anti-clathrin was provided by Abcam (Cambridge, UK). Rabbit monoclonal anti-Sec24B (D7D6S) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal anti-Ac-Histone H3 (Lys 9/14) was ordered from Santa Cruz (Dallas, TX, USA). Rabbit polyclonal anti-actin (A2066) antibodies were purchased from Merck, Inc. (Darmstadt, Germany). Mouse polyclonal anti-PEDV-N and rabbit polyclonal anti-PEDV-N antibodies were previously generated in the lab [3]. The horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG antibodies were ordered from MiliporeSigma (Merck Inc., Darmstadt, Germany). Alexa 488-conjugated goat anti-rabbit IgG (A-11001) and Alexa 555-conjugated goat anti-mouse IgG (A-21428) antibodies were supplied by Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The 10 nm labeled goat anti-rabbit secondary antibody (G7402) was purchased from Sigma-Aldrich (St. Louis, MO, USA). TSA, and NaB were provided by Selleck (Houston, TX, USA). Glutaraldehyde (2.5%, pH 7.4) and paraformaldehyde (4%)-glutaraldehyde (0.5%) mixture, (pH 7.4) were purchased from Yuanye Bio-Technology Co., Ltd., (Shanghai, China). Ethanol was obtained from Sinaopharm Group Chemical Reagent Co. LTD (Shanghai, China). LR White Resin was supplied by HaideBio (Beijing, China). Enhanced chemiluminescence (ECL) reagent was purchased from Youqing Biology (Nanjing, China). 2.3. Viral Infection and Titer Determination PEDV (strain HLJBY) was cultured in Vero-E6 cells. The cells were infected with PEDV in DMEM without FBS and incubated at 37 ◦ C for 1 h. After incubation, the cells were washed with phosphate-buffered saline (PBS) and transferred to DMEM containing 2% FBS and 17.5 ng of trypsin per mL. The virus titer was determined using the plaque formation assay. Briefly, 6-well plates were seeded with 1 × 106 Vero-E6 cells/well the day before inoculation. The cells were washed with PBS, and 10-fold serial dilutions (102 to 107 ) of viruses were incubated with a confluent monolayer of Vero-E6 cells at 37 ◦ C for 1 h. After incubation, the cells were washed with PBS, followed by the addition of 2 mL overlay medium (2% low-melting-point agarose (Lonza Inc., Basel, Switzerland) in 2 × DMEM with 2% FBS and 17.5 ng of trypsin per mL). The plates were incubated at 37 ◦ C with 5% CO2 for 2 to 3 days. The cells were stained with 0.5% crystal violet. 2.4. Western Blotting Analysis Whole-cell extracts were prepared with 2 × SDS sample buffer (4% SDS, 0.1 M Tris HCI pH 6.8, 20% glycerol, 2% bromophenol blue, and 10% β-mercaptoethanol) and boiled for 10 min at 98 ◦ C. Next, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitro-cellulose transfer membrane (Pall Corporation, Port Washington, NY, USA) using a Mighty Small Trans-fer Tank system (Hoefer, MA, USA). Then, the membranes were blocked with 3% non-fat milk in PBS with 0.5% Tween 20 (PBST) for 30 min at room temperature and then incubated with a specific primary antibody overnight at 4 ◦ C followed by incubation with secondary antibodies for 4 h at 4 ◦ C. The positive bands were visualized with the enhanced chemiluminescence (ECL) reagent, and imaged using a BioSpectrum Imaging System (UVP, Upland, CA, USA).
2.5. Knockdown of Sec23A/Sec24B Expression The siRNA oligonucleotides against Sec24B and a nontargeting control siRNA were purchased from Biotend, Inc. (Shanghai, China). For siRNA gene knockdown experiments, 3.6 × 105 Vero-E6 cells were seeded into 6-well plates/well for 18 h and transfected with 50 nM siRNA oligonucleotide using 7.5 µL Mirus following the manufacturer’s instructions (Madison, WI, USA). After 48 h of transfection, the cells were analyzed by immunoblotting to determine the knockdown efficiency. The Sec23A and Sec24B RNA interference (RNAi) target sequences were as follows: siSec23A-1, 50 -AAG GAA UCA GUU UCC ACC UAG UUA U-30 , siSec23A-2,50 -CCU ACA GCU UUG GUU GGA CUU AUU A-30 ; siSec24B-1,50 CGG UAU AUU CUG GAU UCC AAC AGU A-30 , siSec24B-2,50 -CCC GAU CUU AUG GAG AGC CUC AUA A-30 . 2.6. Immunofluorescence Microscopy The Vero-E6 cells were grown on coverslips and pretreated with or without TSA for 2 h, and then infected with PEDV for different durations. The cells on the coverslips were fixed and permeabilized with 4% formaldehyde and 0.1% Triton X-100 at 37 ◦ C for 30 min. After washing with glycine-PBS, the cells were blocked with 3% BSA in PBS at 37 ◦ C for 30 min. The coverslips were incubated with primary antibody (1:200) at 37 ◦ C for 1 h, followed by secondary antibody (1:500) at 37 ◦ C for 30 min. Unbound antibodies were removed by washing with PBST three times. The nuclei were stained with 40 ,6-diamidino2-phenylindole (DAPI) containing the anti-fade Dabco solution (Thermo Fisher Scientific, Waltham, MA, USA). Images of Figures 3 and 4A were obtained with a confocal microscopy (Nikon Eclipse Ti, A1, Tokyo, Japan) and Figure 6C,D and Figure S2 were obtained using a fluorescence microscopy (Nikon Eclipse Ti-U, Tokyo, Japan). 2.7. Transmission Electron Microscopy/Immunoelectron Microscopy Vero-E6 cells were pretreated with or without TSA and NaB for 2 h and then mock infected or infected with PEDV at a MOI = 0.1. After 12 or 24 h, cells were collected and centrifuged into a cell mass. First, the cell mass was fixed in cold glutaraldehyde (2.5%, pH 7.4, TEM) or cold paraformaldehyde (4%)-glutaraldehyde (0.5%) glutaraldehyde mixture, (pH 7.4, IEM) at 4 ◦ C overnight, followed by additional steps including cleaning, fixation, dehydration, resin penetration, embedding, solidification, polymerization, microtomy, and staining. The precipitation was washed three times in 0.1 M PB (pH 7.4), for 10 min each time. It was fixed with 1% osmic acid and then cleaned. The fixed cell mass was gradient dehydrated in different concentrations of ethanol. Resin penetration with different concentrations of embedding agent was performed. An embedding capsule was used for embedding, at 4 ◦ C. The solidification step was performed in a 55◦ C incubator for 48 h for TEM. For more than 48 h, polymerization was conducted at −20 ◦ C with low temperature UV polymerizer-UVCC2515 (Zhongjingkeyi Technology, Beijing, China) for IEM. Ultrathin sections (70–80 nm) from the resin blocks were obtained using a Leica UC7ultramicrotome (Wetzlar, Germany), and the tissues were fished out onto 150-meshes nickel grids (Zhongjingkeyi Technology, Beijing, China) with Formvar film; the sections were stored at 4 ◦ C. Immunolabelling was performed by incubating with an anti-COPII antibody overnight at 4 ◦ C, followed by incubation with 10 nm labeled goat anti-rabbit secondary antibody (20 min at room temperature; 1 h at 37 ◦ C; 30 min at room temperature) for IEM. For TEM/IEM, sample was stained with 2% uranyl acetate and lead citrate. Sections were examined with a Hitachi H-7650 (Hitachi, Tokyo, Japan) TEM at an accelerating voltage of 80 kV. 2.8. Cell Viability Analysis The cytotoxicity of HDAC-specific inhibitors was measured via a Cell Counting Kit8 (CCK-8) assay, following the manufacturer’s instructions (APExBIO Technology, Inc., Houston, TX, USA). In brief, Vero-E6 cells were seeded in the 96-well plate and cultured for 24 h and then treated with an increased dose of HDAC inhibitors for 12 or 24 h, followed
by incubation with CCK-8 reagent (10 µL/well) at 37 ◦ C for 4 h. Finally, the absorbance at 450 nm was measured in an enzyme-linked immunosorbent assay reader. 2.9. Statistical Analysis The results shown are representative of three replicate experiments. All statistical tests were conducted using GraphPad Prism 7.0 software (San Diego, CA, USA) and are presented as means plus or minus standard deviation (SDs). Statistical significance was determined using the Student’s t test. p values of <0.05 were considered statistically significant. 3. Results 3.1. HDAC-Specific Inhibitors Facilitate PEDV Particle Secretion HDAC inhibitors, such as TSA and SAHA, suppress HCV and RSV replication [38–40], whereas romidepsin inhibits the entry of SARS-CoV-2 [41]. This finding prompted us to investigate whether HDAC inhibitors regulate PEDV infection. To verify this possibility, Vero-E6 cells were pretreated with or without TSA and NaB, inhibitors of class I and II HDACs [43,44], and then infected with PEDV. The culture medium and the infected cells were harvested separately. To test the cytotoxicity of the HDAC-specific inhibitors we performed CCK-8 assay. We found that the HDAC-specific inhibitors at concentrations used in this study did not affect the cell viability (Figure 1A,B). To determine the functionality of HDAC-specific inhibitors, the infected cell lysates were analyzed via Western blotting analysis of acetylated histone H3. The protein level of acetylated histone H3 was increased by the HDAC-specific inhibitors treatment (Figure 1C,F). The infected cell lysates and the culture medium were subjected to Western blotting to analyze the intracellular and extracellular PEDV N protein levels. We found that the HDAC-specific inhibitors decreased the intracellular PEDV-N protein levels but increased the extracellular PEDV-N protein levels (Figure 1C,F and Figure S1). Cells treated with TSA secreted about 2.0-fold higher levels of infectious virus particles in the supernatant than the DMSO-treated cells (Figure 1D,E). Cells treated with NaB secreted infectious virus particles in the supernatant about 2.3-fold higher than the control cells (Figure 1G,H). These data suggested that the HDAC-specific inhibitors facilitate the secretion of PEDV virus particles into the extracellular medium. To further analyze the effect of HDAC-specific inhibitors on PEDV infection, Vero-E6 cells were pretreated with or without TSA and NaB and then infected with PEDV. The intracellular and extracellular virus titers were determined by plaque formation assay (PFU). PFU results showed that the HDAC-specific inhibitors treatment decreased the intracellular virus titer and increased the extracellular virus titer (Figure 2A,B,D,E). We used the ratio of virus titers in extracellular and intracellular compartments to evaluate virus release. The results showed that the TSA treatment resulted in an approximately 3.3-fold increase in the release of infectious virus particles (Figure 2C) compared with the approximately 2.7-fold increased by NaB treatment (Figure 2F). However, the total virus titer of HDAC-specific inhibitors-treated cells did not change significantly compared with the control (Figure 2G,H). These data indicated that the HDAC-specific inhibitors promote PEDV release without affecting its assembly.
Figure 1. HDAC-specific promote the promote secretion PEDV of virus particles into the Figure 1. inhibitors HDAC-specific inhibitors theofsecretion PEDV virus particles intoextracelthe extracellulular compartment. (A,B) Determination of the cell viability following treatment with TSA and NaB lar compartment. (A,B) Determination of the cell viability following treatment with TSA and NaB via via CCK-8 test. Vero-E6 cellsVero-E6 were pretreated with or without TSA (40 and and NaBNaB (2 mM) forfor 2 h, CCK-8 test. cells were pretreated with or without TSAng/mL) (40 ng/mL) (2 mM) 2 h, and then mock-infected or infected HLJBY (MOI =(MOI 0.1).=After HLJBY adsorption forfor1 1h.h. The and then mock-infected orwith infected with HLJBY 0.1). After HLJBY adsorption The cells were further cultured fresh medium in theinpresence of TSA and 12or or2424h.h. The cells were furtherin cultured in fresh medium the presence of TSA andNaB NaB at at 12 The infected cellwere lysates were prepared, and AC-H3, intracellular N, actin and actin were detected Western-blot infected cell lysates prepared, and AC-H3, intracellular N, and were detected byby Western(C,F). medium The culture medium wasinto divided twoone parts; onewas partanalyzed was analyzed by Western blotting blot (C,F). The culture was divided twointo parts; part by Western blotof extracellular N (C,F), and the other was titrated by plaque formation assay to measure the ting of extracellular N (C,F), and the other part waspart titrated by plaque formation assay to measure 5 ), 24 h 5),10 extracellular titers (D,G).show Graphs show changes in titers virus titers (E,H); h (× 24 the extracellular PEDV virus PEDV titers virus (D,G). Graphs changes in virus (E,H); 12 h12(×10 6 ). Student’s t test was used for statistical analysis. **, p < 0.01; ***, p < 0.001. The error bars was used for statistical analysis. **, p < 0.01; ***, p < 0.001. The error bars h (×106). Student’s(×t10test standard from three independent experiments. In; intracellular, Ex; extracellular. indicate standardindicate deviation from deviation three independent experiments. In; intracellular, Ex; extracellular.
To further analyze the effect of HDAC-specific inhibitors on PEDV infection, VeroE6 cells were pretreated with or without TSA and NaB and then infected with PEDV. The intracellular and extracellular virus titers were determined by plaque formation assay (PFU). PFU results showed that the HDAC-specific inhibitors treatment decreased the intracellular virus titer and increased the extracellular virus titer (Figure 2A,B,D,E). We used the ratio of virus titers in extracellular and intracellular compartments to evaluate virus
HDAC-specific inhibitors-treated cells did not change significantly compared with the control (Figure 2G,H). These data indicated that the HDAC-specific inhibitors promote PEDV release without affecting its assembly.
7 of 16
Figure
2. HDAC-specific
inhibitors
facilitatefacilitate
the secretion
of infectious
particles.
Vero-E6
cells Vero-E6 cells
Figure
2. HDAC-specific
inhibitors
the secretion
of PEDV
infectious
PEDV
particles.
were pretreated with or without TSA (40 ng/mL) and NaB (2 mM) for 2 h, and then mock-infected
were pretreated with or without TSA (40 ng/mL) and NaB (2 mM) for 2 h, and then mock-infected or
or infected with HLJBY (MOI = 0.1). After HLJBY adsorption for 1 h, the cells were further cultured
infected
withinHLJBY
(MOI of
= 0.1).
After
HLJBY
adsorption
for 1 h,medium
the cellswas
were
further
in fresh
medium
the presence
TSA and
NaB
at 12 or
24 h. The culture
first
col- cultured in
lected,
and
the
infected
cells
were
washed
thrice
with
phosphate-buffered
saline
(PBS)
before
addfresh medium in the presence of TSA and NaB at 12 or 24 h. The culture medium was first collected,
ing and
freshthe
medium,
and
then
lysed
via three
freeze/thaw
cycles to obtain the saline
intracellular
infected
cells
were
washed
thrice
with phosphate-buffered
(PBS) PEDV.
before adding fresh
(A,D): The virus titers (intracellular and extracellular) were detected by plaque formation assay.
medium, and then lysed via three freeze/thaw cycles to obtain the intracellular
PEDV. (A,D): The
(B,E): Graphs show changes in virus titers (intracellular and extracellular); 12 h (×105), 24 h (×106).
virus
titers
(intracellular
and
extracellular)
were
detected
by
plaque
formation
assay.
(C,F): Graphs how PEDV release (ratio of extracellular to intracellular virus titers). (G,H): Graphs (B,E): Graphs
6 ). (C,F): Graphs
show
changes
in total
virus titers
titer. Student’s
t test and
was extracellular);
used for statistical
show
changes
in virus
(intracellular
12 hanalysis.
(×105 ),The
24 herror
(×10bars
indicate
standard
deviation
from
three
independent
experiments.
ns,
p
>
0.05;
*,
p
<
0.05;
***,
p in total virus titer. Student’s t test was used for statistical analysis. The error bars indicate standard
deviation from three independent experiments. ns, p > 0.05; *, p < 0.05; ***, p < 0.001. 3.2. Colocalization of PEDV N Proteins and Its Replication Complex with COPII-Coated Vesicles
It has been reported that PEDV utilizes smooth-walled vesicles to egress [14]. Previous
studies have shown that intracellular trafficking vesicles in eukaryotic cells are mainly
composed of COPI, COPII and CCVs [17,18]. To determine the vesicular transport involved
in PEDV transport, Vero-E6 cells were infected with PEDV. At different time points, VeroE6 cells were stained with anti-Sec21p (γ subunit of COPI), anti-COPII and anti-clathrin
antibodies to detect transport vesicles. The subcellular localization of PEDV N protein
and trafficking vesicle markers was examined using a confocal microscopy. The confocal
microscopic images showed that only COPII colocalized with PEDV N protein and the It has been reported that PEDV utilizes smooth-walled vesicles to egress [14]. Previous studies have shown that intracellular trafficking vesicles in eukaryotic cells are mainly
composed of COPI, COPII and CCVs [17,18]. To determine the vesicular transport involved in PEDV transport, Vero-E6 cells were infected with PEDV. At different time
points, Vero-E6 cells were stained with anti-Sec21p (γ subunit of COPI), anti-COPII and
8 of 16
anti-clathrin antibodies to detect transport vesicles. The subcellular localization of PEDV
N protein and trafficking vesicle markers was examined using a confocal microscopy. The
confocal microscopic images showed that only COPII colocalized with PEDV N protein
PEDV
infection
enhanced
the colocalization
of COPIIofand
PEDV
in a time-dependent
and the
PEDV infection
enhanced
the colocalization
COPII
andNPEDV
N in a time-demanner
Neither
nor
clathrin
colocalized
with PEDV
protein
at the
pendent(Figure
manner3B).
(Figure
3B). COPI
Neither
COPI
nor clathrin
colocalized
with N
PEDV
N protein
indicated
time points
3A,C). Vero-E6
cells were
stained
with anti-dsRNA
at the indicated
time (Figure
points (Figure
3A,C). Vero-E6
cellsalso
were
also stained
with antiantibody
to detecttothe
virus
replication
complex.
We found
that COPII
colocalized
with
dsRNA antibody
detect
the
virus replication
complex.
We found
that COPII
colocalized
PEDV
replicative
intermediate
dsRNA
(Figure
3D).
These
findings
suggested
that
COPIIwith PEDV replicative intermediate dsRNA (Figure 3D). These findings suggested that
coated
vesiclesvesicles
mediatemediate
PEDV trafficking.
COPII-coated
PEDV trafficking. Figure3.3. PEDV
PEDV N
N protein
protein and
and its
itsreplicative
replicative intermediate
intermediate dsRNA
dsRNA colocalize
colocalize with
with COPII-coated
COPII-coated
Figure
vesicles.
Vero-E6
cells
infected
with
HLJBY
(MOI
=
0.1)
for
0,
6,
12
and
24
h.
The
cells
were
fixedfixed
and
vesicles. Vero-E6 cells infected with HLJBY (MOI = 0.1) for 0, 6, 12 and 24 h. The cells were
stained with Sec21p (γ subunit of COPI) antibody (A); COPII antibody (B); clathrin antibody (C);
and stained with Sec21p (γ subunit of COPI) antibody (A); COPII antibody (B); clathrin antibody
and Alexa 488-conjugated goat anti-rabbit IgG antibodies (green) and then stained with PEDV-N
(C); and Alexa 488-conjugated goat anti-rabbit IgG antibodies (green) and then stained with PEDV-N
antibody and Alexa 555-conjugated goat anti-mouse IgG antibody (red). The nuclei were stained
antibody
and
AlexaImages
555-conjugated
goat with
anti-mouse
antibody
(red). The
nucleipanel
were shows
staineda
with DAPI
(blue).
were acquired
a NikonIgG
confocal
microscopy.
Bottom
with DAPI (blue). Images were acquired with a Nikon confocal microscopy. Bottom panel shows a
magnified view of the boxed area in panels (A–C) (Merged). For quantitative colocalization analysis
(QCA), Pearson correlation coefficient (PCC) values were calculated and represent the mean ± SD.
(D) Vero-E6 cells infected with HLJBY (MOI = 0.1) for 24 h. The cells were fixed and stained with
COPII antibody and Alexa 488-conjugated goat anti-rabbit IgG anti-bodies (green), and then stained
with dsRNA antibody and Alexa 555-conjugated goat anti-mouse IgG antibody (red). The nuclei
were stained with DAPI (blue). Images were acquired with a Nikon confocal microscopy. Bottom
panel shows a magnified view of the boxed area in panel (D) (Merge). For quantitative colocalization
analysis (QCA), PCC values were calculated and represent the mean ± SD. SD. (D) Vero-E6 cells infected with HLJBY (MOI = 0.1) for 24 h. The cells were fixed and stained with
COPII antibody and Alexa 488-conjugated goat anti-rabbit IgG anti-bodies (green), and then stained
with dsRNA antibody and Alexa 555-conjugated goat anti-mouse IgG antibody (red). The nuclei
were stained with DAPI (blue). Images were acquired with a Nikon confocal microscopy. Bottom
panel shows a magnified view of the boxed area in panel D (Merge). For quantitative colocalization
analysis (QCA), PCC values were calculated and represent the mean ± SD.
9 of 16 3.3. PEDV N Protein Is Efficiently Captured by The COPII-Coated Vesicles upon HDACSpecific Inhibitors Treatment
3.3. PEDV N Protein Is Efficiently Captured by The COPII-Coated Vesicles upon HDAC-Specific
Our Treatment
study showed that the HDAC-specific inhibitors facilitated PEDV release (FigInhibitors
ures Our
1, 2 and
S1) showed
and COPII
was
colocalized
with the
PEDV N facilitated
protein (Figure
To destudy
that
the
HDAC-specific
inhibitors
PEDV3).release
termine
whether
HDAC-specific
inhibitors
promote
the
colocalization
of
COPII
(Figures 1, 2 and S1) and COPII was colocalized with the PEDV N protein (Figure 3).and
To
PEDV N protein,
cells were
infected
with the
PEDV
with or without
TSA
NaB
determine
whether Vero-E6
HDAC-specific
inhibitors
promote
colocalization
of COPII
andorPEDV
treatment.
Vero-E6 cells
cellswere
wereinfected
stained with
to TSA
identify
transport
vesN
protein, Vero-E6
with anti-COPII
PEDV with antibody
or without
or NaB
treatment.
icles.
The
colocalization
of
COPII
and
PEDV
N
was
analyzed
with
confocal
microscopy.
Vero-E6 cells were stained with anti-COPII antibody to identify transport vesicles. The colocalPCC values
showed
that the
of COPII
andmicroscopy.
PEDV N protein
was enhanced
ization
of COPII
and PEDV
N colocalization
was analyzed with
confocal
PCC values
showed
by
HDAC-specific
inhibitors
(Figure
4A,B).
These
findings
indicated
that
the
HDAC-spethat the colocalization of COPII and PEDV N protein was enhanced by HDAC-specific
incific inhibitors
PEDV
N proteins
capture
the COPII-coated
vesicles.
Immuhibitors
(Figure promote
4A,B). These
findings
indicated
thatinto
the HDAC-specific
inhibitors
promote
nofluorescence
inhibitors
reduced PEDV N protein
PEDV
N proteinshowed
capturethat
intothe
theHDAC-specific
COPII-coated vesicles.
Immunofluorescence
showedlevels
that
(Figure
S2).
the
HDAC-specific
inhibitors reduced PEDV N protein levels (Figure S2). Figure 4.
4. The
The colocalization between PEDV N and COPII is enhanced by HDAC-specific inhibitors.
Figure
Vero-E6 cells
cells were
were pretreated
pretreatedwith
withor
orwithout
withoutTSA
TSA(60
(60ng/mL)
ng/mL) and
NaB (4
(4 mM)
mM) for
for 22 h,
h, and
and then
then
Vero-E6
and NaB
infected
with
HLJBY
(MOI
=
1)
for
1
h.
The
cells
were
further
cultured
in
fresh
medium
in
the
presinfected with HLJBY (MOI = 1) for 1 h. The cells were further cultured in fresh medium in the
ence of TSA and NaB at 8 h. (A) The cells were fixed and stained with COPII antibody and Alexa
presence of TSA and NaB at 8 h. (A) The cells were fixed and stained with COPII antibody and Alexa
488-conjugated goat anti-rabbit IgG antibodies (green) and then stained with PEDV-N antibody and
488-conjugated goat anti-rabbit IgG antibodies (green) and then stained with PEDV-N antibody and
Alexa 555-conjugated goat anti-mouse IgG antibody (red). The nuclei were stained with DAPI
Alexa
goatacquired
anti-mouse
antibody
(red). microscopy.
The nuclei were
(blue).
(blue).555-conjugated
The images were
withIgG
a Nikon
confocal
(B) stained
Graphs with
showDAPI
the PCC
of
The images were acquired with a Nikon confocal microscopy. (B) Graphs show the PCC of COPII and
PEDV N. For quantitative colocalization analysis (QCA), PCC values were calculated and represent
the mean ± SD. Student’s t test was used for statistical analysis. ***, p < 0.001; ****, p < 0.0001. 3.4. Secretion of COPII-Coated Vesicles Carrying PEDV Virions Is Promoted by
HDAC-Specific Inhibitors
To establish the PEDV secretion induced by HDAC-specific inhibitors, we analyzed
the inhibitory effect on PEDV compartmentation using TEM. Vero-E6 cells were infected
with PEDV and treated with or without TSA and NaB. The PEDV virus particles were
detected by TEM. As expected, it was found that PEDV virions mainly accumulate in 0.0001. 3.4. Secretion of COPII-Coated Vesicles Carrying PEDV Virions Is Promoted by HDAC-Specific
Inhibitors
Viruses 2023, 15, 1874 To establish the PEDV secretion induced by HDAC-specific inhibitors, we analyzed 10 of 16
the inhibitory effect on PEDV compartmentation using TEM. Vero-E6 cells were infected
with PEDV and treated with or without TSA and NaB. The PEDV virus particles were
detected by TEM. As expected, it was found that PEDV virions mainly accumulate in ERER-like
luminal
structures
in control
In contrast,
virions
the HDAC-specific
like luminal
structures
in control
cells.cells.
In contrast,
PEDVPEDV
virions
in thein
HDAC-specific
inhibitors-treated
cells were
weremainly
mainlyenclosed
enclosedwithin
withintransport
transport
vesicles;
arrows
indicate
inhibitors-treated cells
vesicles;
thethe
arrows
indicate some
that some
transport
vesicles
containingonly
only one virion
containing
multithat
transport
vesicles
containing
virionand
andothers
others
containing
multiple
ple virions
(Figure
5A–D).
These
findings
suggested
HDAC-specific
inhibitors
virions
(Figure
5A–D).
These
findings
suggested
thatthat
the the
HDAC-specific
inhibitors
promote
promote
the secretion
of transport
vesicles containing
PEDV virions.
Intorder
to whether
verify
the
secretion
of transport
vesicles containing
PEDV virions.
In order
verify
the
whether he
transport
vesicles
were COPII-coated
we performed
to analyze
transport
vesicles
were
COPII-coated
vesicles, vesicles,
we performed
IEM toIEM
analyze
the COPIIthe COPII-coated
PEDVPEDV
virions.
PEDV
was
to be enclosed
within
coated
vesicles forvesicles
PEDV for
virions.
was
found
to found
be enclosed
within COPII-coated
COPII-coated
vesicles
(Figure
5E).
The
data
showed
that
the
HDAC-specific
inhibitors
vesicles (Figure 5E). The data showed that the HDAC-specific inhibitors promote the
promote the
secretion of COPII-coated
vesiclesPEDV
carrying
PEDV virions.
secretion
of COPII-coated
vesicles carrying
virions. Figure 5. PEDV virions localized to COPII-coated vesicles following HDAC-specific inhibitors treatFigure
5. PEDV virions localized to COPII-coated vesicles following HDAC-specific inhibitors
ment. Vero-E6 cells were pretreated with or without TSA (60 ng/mL) and NaB (4 mM) for 2 h, and
treatment.
Vero-E6 cells were pretreated with or without TSA (60 ng/mL) and NaB (4 mM) for
then mock infected or infected with HLJBY (MOI = 0.1). After HLJBY adsorption for 1 h, the cells
2were
h, and
then
mock infected
infected
with
HLJBYof(MOI
= 0.1).
further cultured
in fresh or
medium
in the
presence
TSA and
NaBAfter
at 12 HLJBY
or 24 h. adsorption
(A,C): Virusfor 1 h,
particles
a transmission
electron microscopy
(TEM).of
Arrows
indicate
transport
the
cells were
weredetected
further with
cultured
in fresh medium
in the presence
TSA and
NaB
at 12 or 24 h.
vesicles
containing
virions.
Graphs
the percentage
of vesicles
carrying
PEDV
virions.indicate
(A,C):
Virus
particles
were (B,D):
detected
with show
a transmission
electron
microscopy
(TEM).
Arrows
Student’s t test was used for statistical analysis. ***, p < 0.001. The error bars indicate standard devitransport vesicles containing virions. (B,D): Graphs show the percentage of vesicles carrying PEDV
ation from three independent experiments. (E): Detection of COPII-coated vesicles by immunoelecvirions.
Student’s
t test
was
for statistical
analysis.
***, p 3.5. HDAC-Specific Inhibitor-Promoted Secretion of PEDV Is Dependent on COPII Complex
The COPII complex consists of five subunits: Sar1, Sec23, Sec24, Sec13 and Sec31 [23].
Sec23 acts as a GAP for Sar1. Sec24 is a primary subunit of the COPII-coated vesicles,
which is responsible for the transportation of cargos from the ER to the Golgi apparatus [23,45,46]. A schematic diagram of the COPII-coated vesicles formation is presented
in Figure 6A. To further establish the correlation between the COPII-coated vesicles and
virus release, we disrupted the formation of the COPII-coated vesicles by knocking down
Sec23A or Sec24B. Vero-E6 cells were transfected with Sec23A or Sec24B small interfering
RNA (siRNA), followed by infection with PEDV with or without TSA treatment. The 3.5. HDAC-Specific Inhibitor-Promoted Secretion of PEDV Is Dependent on COPII Complex Viruses 2023, 15, 1874 The COPII complex consists of five subunits: Sar1, Sec23, Sec24, Sec13 and Sec31 [23].
Sec23 acts as a GAP for Sar1. Sec24 is a primary subunit of the COPII-coated vesicles,
which is responsible for the transportation of cargos from the ER to the Golgi apparatus
[23,45,46]. A schematic diagram of the COPII-coated vesicles formation is presented in
11 of 16
Figure 6A. To further establish the correlation between the COPII-coated vesicles and virus release, we disrupted the formation of the COPII-coated vesicles by knocking down
Sec23A or Sec24B. Vero-E6 cells were transfected with Sec23A or Sec24B small interfering
RNA (siRNA),levels
followed
by infection
PEDVwere
with significantly
or without TSA
treatment.in
The
en- treated
endogenous
of Sec23A
andwith
Sec24B
decreased
cells
dogenous
levels of Sec23A and Sec24B
significantly
in cells treated
with
with
Sec23A/Sec24B-specific
siRNAwere
compared
with decreased
cells transfected
with nontargeting
Sec23A/Sec24B-specific
siRNA
with cells transfected
scrambled
siRNA (Figure
6B).compared
Immunofluorescence
showedwith
thatnontargeting
TSA reducedscramthe PEDV N
bled
siRNA
(Figure
6B).
Immunofluorescence
showed
that
TSA
reduced
PEDV
N level had no
level in the cells transfected with non-targeting siRNA. However,the
TSA
treatment
in the cells transfected
with non-targeting
siRNA.
However,
treatment
had no signif- siRNA
significant
effect on PEDV
N levels in cells
treated
withTSA
Sec23A
or Sec24B-specific
icant effect on PEDV N levels in cells treated with Sec23A or Sec24B-specific siRNA (Fig(Figure 6C,D). The PFU data showed that TSA decreased the intracellular virus titer and
ure 6C,D). The PFU data showed that TSA decreased the intracellular virus titer and inincreased the extracellular virus titer in cells with non-targeting siRNA transfected cells
creased the extracellular virus titer in cells with non-targeting siRNA transfected cells
(Figure
6E). PEDV release was evaluated using the ratio of extracellular-to-intracellular
(Figure 6E). PEDV release was evaluated using the ratio of extracellular-to-intracellular
virus
titers.
Wefound
foundthat
that
PEDV
release
by TSA
was inhibited
cellsSec24B
with Sec24B
virus titers. We
PEDV
release
by TSA
was inhibited
in cellsinwith
knock- knockdown
cells
(Figure
6F).
The
above
results
indicated
that
Sec24B-containing
COPII-coated
down cells (Figure 6F). The above results indicated that Sec24B-containing COPII-coated
vesicles
are essential
essentialfor
forPEDV
PEDVrelease
release
following
HDAC-specific
inhibitors
treatment.
vesicles are
following
HDAC-specific
inhibitors
treatment.
In- Interestingly,
we
found
that
the
decrease
in
PEDV
N
protein
levels
induced
by
HDAC-specific
terestingly, we found that the decrease PEDV N protein levels induced by HDAC-speinhibitors
leads
to atodecrease
in in
Sec23A/Sec24B
levels(Figure
(Figure
S3).
cific inhibitors
leads
a decrease
Sec23A/Sec24B protein
protein levels
S3). Figure 6. Suppression of HDAC-specific inhibitors-induced PEDV secretion by Sec24B knockdown.
(A): Schematic diagram of the vesicle budding process in the COPII complex. Vero-E6 cells were
transfected with nontargeting scrambled siRNA or targeting Sec23A/Sec24B siRNA for 48 h, and then
pretreated with or without TSA (40 ng/mL) for 2 h, followed by HLJBY (MOI = 0.1) infection. After
HLJBY adsorption for 1 h, the cells were further cultured in fresh medium in the presence of TSA for
12 h. (B): Sec23A/Sec24B and actin were detected by immunoblotting. (C,D): The cells were fixed
and stained with Sec24B or COPII antibody and Alexa 488-conjugated goat anti-rabbit IgG antibodies
(green) and then stained with PEDV-N antibody and Alexa 555-conjugated goat anti-mouse IgG
antibody (red). The nuclei were stained with DAPI (blue). The images were acquired with a Nikon
immunofluorescence microscopy. (E): Virus titers were measured via plaque formation assay, and
the graphs show changes in virus titers (intracellular and extracellular); 12 h (×105 ). (F): Graph
shows PEDV release (ratio of extracellular to intracellular virus titers). Student’s t test was used for
statistical analysis. ***, p < 0.001; ****, p < 0.0001. The error bars indicate standard deviation from
three independent experiments. antibodies (green) and then stained with PEDV-N antibody and Alexa 555-conjugated goat antimouse IgG antibody (red). The nuclei were stained with DAPI (blue). The images were acquired
with a Nikon immunofluorescence microscopy. (E): Virus titers were measured via plaque formation assay, and the graphs show changes in virus titers (intracellular and extracellular); 12 h
(×105). (F): Graph shows PEDV release (ratio of extracellular to intracellular virus titers). Student’s t
12 of 16
test was used for statistical analysis. ***, p < 0.001; ****, p < 0.0001. The error bars indicate standard
deviation from three independent experiments. 4. Discussion
Discussion
4.
PED is a highly
inin
pigs.
PEDV
infection
results
in high
morPED
highlycontagious
contagiousviral
viraldisease
disease
pigs.
PEDV
infection
results
in high
tality in neonatal
suckling
piglets
[47,48].
Similar
to other
RNA viruses,
PEDVs
mortality
in neonatal
suckling
piglets
[47,48].
Similar
to enveloped
other enveloped
RNA viruses,
uses and
modifies
intracellular
compartments
of the secretory
pathwaypathway
to facilitate
viral
PEDVs
uses
and modifies
intracellular
compartments
of the secretory
to facilireplication
and egression
[13]. However,
the underlying
mechanisms
of PEDV of
secretion
tate
viral replication
and egression
[13]. However,
the underlying
mechanisms
PEDV
secretion
remainunknown.
largely unknown.
In thiswe
study,
we demonstrated
the HDAC-specific
remain largely
In this study,
demonstrated
that thethat
HDAC-specific
inhibiinhibitors
the COPII-coated
vesicles
to promote
7). HDAC
tors hijackhijack
the COPII-coated
vesicles
to promote
PEDVPEDV
releaserelease
(Figure(Figure
7). HDAC
inhibiinhibitors
have
been
reported
to
induce
antiviral
effects
against
a
few
enveloped
RNA
tors have been reported to induce antiviral effects against a few enveloped RNA viruses,
viruses,
including
RSV,
HCV
and SARS-CoV-2.
TSA and
canRSV
suppress
RSVrepliand
including
RSV, HCV
and
SARS-CoV2.
TSA and SAHA
canSAHA
suppress
and HCV
HCV
[38–40]. Romidepsin
of SARS-CoV-2
In the present
cationreplication
[38–40]. Romidepsin
preventsprevents
the entrythe
ofentry
SARS-CoV2
[41]. In [41].
the present
study,
study,
we found
that
TSA
and
NaB promote
the secretion
PEDV
virus into
particles
into
we found
that TSA
and
NaB
promote
the secretion
of PEDVof
virus
particles
the extrathe
extracellular
compartment
andConsistently,
S1). Consistently,
the PFU
showed
cellular
compartment
(Figures(Figures
1 and 1S1).
the PFU
datadata
showed
thatthat
the
the
HDAC-specific
inhibitors
facilitate
the
secretion
but
not
assembly
of
infectious
PEDV
HDAC-specific inhibitors facilitate the secretion but not assembly of infectious PEDV parparticles
(Figure
ticles (Figure
2). 2). Figure 7. The interplay between HDAC-specific inhibitors and COPII-coated vesicles during PEDV
Figure 7. The interplay between HDAC-specific inhibitors and COPII-coated vesicles during PEDV
infection.
infection. Following
Following the
the entry
entry of
of PEDV
PEDV into
into host
host cells
cells and
and replication
replication in
in the
thecytoplasm,
cytoplasm, the
thenewly
newly
synthesized
PEDV
viral
components
and
PEDV
virus
particles
are
transported
to
Golgi
apparatus
synthesized PEDV viral components and PEDV virus particles are transported to Golgi apparatus
by
by the
theCOPII-coated
COPII-coatedvesicles.
vesicles. HDAC-specific
HDAC-specific inhibitors
inhibitors facilitate
facilitate PEDV
PEDV release
release by
by promoting
promoting the
the
secretionof
ofCOPII-coated
COPII-coatedvesicles
vesiclescarrying
carryingPEDV
PEDVvirions.
virions.
secretion Virion assembly occurs in the cytoplasmic side of the ERGIC/Golgi compartments
in CoVs. Alpha-coronaviruses (TGEV, HCoV-NL36) and beta-coronaviruses (MHV) can
assemble in the ER at later stages of virus infection [8–12]. PEDV has been reported to
assemble in both the ER and Golgi apparatus, and the newly assembled virus particles
subsequently utilize the secretory pathway for egression [13]. In our study, we observed
the accumulation of PEDV virions mainly in the expanded ER lumen of control cells. In
contrast, PEDV virions were mainly enclosed within transport vesicles in the HDACspecific inhibitors-treated cells (Figure 5A–D). Further, we found that COPII-coated vesicles
carried PEDV virions (Figure 5E). Thus, the results indicated that the HDAC-specific
inhibitors promoted the secretion of COPII-coated vesicles carrying PEDV virions. HDACspecific inhibitors promote the release of PEDV, and the virus could utilize the COPII-coated
vesicles for intracellular trafficking. Vesicle transport requires a group of conserved proteins,
such as Rab GTPases, motor adaptors, and motor proteins to ensure vesicle transport along
the cytoskeletal track [17]. HDAC inhibitors are able to affect cell transport, the promotion of IAV virion release by tubacin via acetylated microtubules [49,50]. Therefore, whether
TSA and NaB promote the movement of the COPII-coated vesicles carrying PEDV virions
along cytoskeletal remains to be determined.
COPII-coated vesicles promote the intracellular transport of cargos from the ER to
the Golgi apparatus. COPII-coated vesicles have been reported to mediate in intracellular transport of several viruses, such as Ebola virus, Marburg virus, hepatitis B virus,
HCV, parvovirus, and rotavirus [30–34]. Here, we demonstrated that the HDAC-specific
inhibitors-induced COPII-coated vesicles are essential for PEDV release (Figure 6). These
findings also suggest that PEDV assembly is inhibited by Sec23A or Sec24B depletion
(Figure 6). This indicated that the COPII-coated vesicles not only mediate the transport of
PEDV virion assembled in the ER, but also mediate the transport of PEDV viral protein
from the ER to Golgi for assembly. Our study is consistent with previous studies reporting
that PEDV particles can be assembled in both the ER and Golgi apparatus [13].
It is generally assumed that virions that assemble in the ER are exported via the
COPII-mediated early secretory pathway; the virions first reach the Golgi apparatus and
TGN, followed by transport to the plasma membrane and egress [8]. However, a few
studies reported that the beta-coronavirus MHV use lysosomes for egress and that the
enteroviruses (poliovirus) and classical swine fever virus (CSFV) release occurred via
autophagy pathway [10,51,52]. Whether PEDV viral particles in COPII-coated vesicles may
use the biosynthetic secretory pathway or autophagosomes/lysosomes to egress requires
further investigation. In summary, our findings reveal that PEDV virions assemble in
the ER and bud off from the ER in the COPII-coated vesicles. We provide clear evidence
to show that the HDAC-specific inhibitors hijack the COPII-coated vesicles to promote
PEDV release.