African Swine Fever Virus K205R Induces ER Stress and Consequently Activates Autophagy and the NF-κB Signaling Pathway

African swine fever virus (ASFV) is responsible for enormous economic losses in the global swine industry. The ASFV genome encodes approximate 160 proteins, most of whose functions remain largely unknown. In this study, we examined the roles of ASFV K205R in endoplasmic reticulum (ER) stress, autophagy, and inflammation. We observed that K205R was located in both the cytosolic and membrane fractions, and formed stress granules in cells. Furthermore, K205R triggered ER stress and activated the unfolded protein response through activating the transcription factor 6, ER to nucleus signaling 1, and eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3/PERK) signaling pathways. Moreover, K205R inhibited the serine/threonine kinase 1 and the mechanistic target of the rapamycin kinase signaling pathway, thereby activating unc-51 like autophagy activating kinase 1, and hence autophagy. In addition, K205R stimulated the translocation of P65 into the nucleus and the subsequent activation of the nuclear factor kappa B (NF-κB) signaling pathway. Inhibition of ER stress with a PERK inhibitor attenuated K205R-induced autophagy and NF-κB activation. Our data demonstrated a previously uncharacterized role of ASFV K205R in ER stress, autophagy, and the NF-κB signaling pathway.


Introduction
African swine fever virus (ASF), the only known DNA arbovirus, is prevalent in Africa, Europe, and Asia [1]. It causes a highly infectious and fatal hemorrhagic disease affecting the global swine industry [2]. Its genome contains approximately 160 major open reading frames (ORFs) and encoding products, including enzymes, structural proteins, and scaffolding proteins [3]. Some ASFV ORFs have been shown to be involved in regulating viral replication and host antiviral responses. For instance, ASFV A224L, A179L, EP153R, and DP71L inhibit apoptosis and prevent premature cell death, while ASFV E199L induces mitochondria-dependent apoptosis, supporting viral replication [4,5]. In addition, ASFV MGF-505-7R and MGF-505-11R negatively regulate the stimulator of IFN genes (STING)dependent antiviral responses [6,7]. However, the function of most ASFV ORFs remains largely unknown. Given the lack of an effective vaccine against ASFV, it would be of great

Plasmids and Transfection
The coding sequence of ASFV K205R was synthesized by GenScript and cloned into pcDNA3.1 (Invitrogen, Waltham, MA, USA) fused with an HA tag sequence or into pEGFP-C1 (Clontech, Mountain View, CA, USA). All plasmids were transfected with Lipofectamine 3000 (Invitrogen), according to the manufacturer's instructions.

Cell Viability Assays
The HeLa cells were seeded in 60-mm dishes at a density of 4 × 10 5 cells per dish and transfected with pcDNA3.1 (vector, 5 µg) and K205R-HA plasmid (5 µg) for 24 and 48 h, respectively. The cell viability was determined with a CCK-8 cell counting assay kit (DingGuo, Beijing, China), according to the manufacturer's instructions.

Immunoblotting Analysis
The cells were seeded in 60-mm dishes at a density of 7 × 10 5 cells per dish and transfected with indicated plasmids (0-5 µg) for 24 h. The cells were collected in RIPA buffer (Solarbio, Beijing, China) supplemented with protease and phosphatase inhibitor cocktail (MedChemExpress). The protein concentrations of the lysates were quantified with a BCA Protein Assay Kit (DingGuo). The protein samples were separated by SDS-PAGE and transferred to membranes (Millipore, Billerica, MA, USA), which were incubated in 5% nonfat milk (Sangon) at room temperature for 1 h afterwards. The membranes were incubated with primary antibodies at 4 • C overnight and then incubated with horseradish-peroxidaseconjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h. The immunoblotting results were visualized with Luminata Crescendo Western HRP Substrate (Millipore) on a GE AI600 imaging system. The densitometric analysis of target proteins was performed with the ImageJ software (https://imagej.nih.gov/ij/ accessed on 27 September 2021).

Separation of the Soluble and Insoluble Fractions from Cells
The cells were seeded in 60-mm dishes at a density of 7 × 10 5 cells per dish and transfected with indicated plasmids (5 µg) for 24 h. For separation of soluble and insoluble fractions, the cells were lysed in RIPA buffer (Solarbio) supplemented with protease and phosphatase inhibitor cocktail (MedChemExpress), and centrifuged at 12,000× g at 4 • C for 15 min. The supernatant was considered the soluble fraction, whereas the insoluble pellet was directly mixed with 1 × NuPage LDS Sample Buffer (Invitrogen) and heated at 100 • C for 30 min. The extracted fractions were subjected to immunoblotting analysis.

Nuclear and Cytoplasmic Extraction from Cells
The cells were seeded in 60-mm dishes at a density of 7 × 10 5 cells per dish and transfected with indicated plasmids (5 µg) for 24 h. Nuclear and cytoplasmic extraction was performed with NEPER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. The extracted fractions were subjected to immunoblotting analysis.

Separation of Cytosolic and Membrane Fractions from Cells
The cells were seeded in 60-mm dishes at a density of 7 × 10 5 cells per dish and transfected with indicated plasmids (5 µg) for 24 h. Briefly, the cells were homogenized in 0.5 mL of homogenization buffer (10 mM HEPES pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 5 mM sodium EDTA, 5 mM sodium EGTA, and 250 mM sucrose) supplemented with protease and phosphatase inhibitors (MedChemExpress), then centrifuged at 1000× g at 4 • C for 7 min. The pellet, containing the crude nuclear fraction, was discarded, and the supernatant was centrifuged at 12,000× g at 4 • C for 15 min. The resulting supernatant comprised the cytosol, and the pellet containing the membrane fraction was dissolved in lysis buffer (10 mM Tris HCl pH 6.8, 100 mM NaCl, 1% SDS, 1 mM EDTA, and 1 mM EGTA) supplemented with protease and phosphatase inhibitors. The whole cell lysate, cytosolic, and membrane samples were mixed individually with 4 × SDS loading buffer, boiled at 95 • C for 5 min, and subjected to SDS-PAGE and immunoblotting analysis.

The qRT-PCR
The cells were seeded in 12-well plates at a density of 3 × 10 5 cells per dish and transfected with indicated plasmids (2 µg) for 24 h. Total RNA was isolated with TRIzol Reagent (TaKaRa, Shiga, Japan) and subjected to cDNA synthesis with a PrimeScript™ RT reagent Kit (TaKaRa). qRT-PCR was performed in triplicate with SYBR Premix Ex Taq (TaKaRa), according to the manufacturer's instructions. The data were normalized to the level of β-actin expression in each individual sample. The melting curve analysis indicated formation of a single product in all cases. The 2 −∆∆Ct method was used to calculate relative expression changes. The primers used for qRT-PCR were as follows: β-actin-Fw:

Immunofluorescence Analysis
The cells were seeded in 12-well plates with coverslips at a density of 3 × 10 5 cells per dish and transfected with indicated plasmids (2 µg) for 24 h. The cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and were then washed three times with PBS. The cells were permeabilized in PBS containing 0.1% Triton X-100 and blocked with 10% FBS in PBS. The primary antibodies were diluted with 10% FBS in PBS and incubated with the cells for 1 h at room temperature. After being washed with PBS, the cells were incubated with Alexa Fluor 568 goat anti-mouse IgG or Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature. The cells were finally washed in PBS and mounted in ProLong Diamond with DAPI (Invitrogen). Images were captured with a Zeiss LSM 800 confocal microscope.

ELISA
The cells were seeded in 12-well plates with coverslips at a density of 3 × 10 5 cells per dish and transfected with indicated plasmids (2 µg) for 24 h. Concentrations of porcine IL-18 were measured in the cell supernatants with ELISA kits (Advanced BioChemical, Lawrenceville, GA, USA), according to the manufacturer's instructions.

Statistical Analysis
All data were obtained from three independent experiments for quantitative analyses and are expressed as means ± standard errors. All data were analyzed in Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA) with two-tailed Student's t-tests, and p < 0.05 was considered statistically significant.

Subcellular Localization of ASFV K205R
To better understand K205R's function, we constructed a plasmid for the expression of K205R fused with an HA tag. We transfected the plasmid into the 3D4/21 and HeLa cells and detected its expression by immunoblotting, which confirmed the expression of  (Figure 2A). Furthermore, CCK-8 cell counting assay showed that K205R-HA resulted in lower cell viability than that of control cells ( Figure 2B), indicating that K205R might affect cell viability.

ASFV K205R Activates ER Stress
Cellular stress, such as ER stress, is a strong inducer of SG formation [24]. Therefore, we sought to determine whether K205R induces ER stress. There are three ER stress sensor pathways, IRE1, PERK and ATF6, which are critical to maintain ER homeostasis [25]. Using immunoblotting analysis, we detected that the expression of K205R in 3D4/21 cells stimulated ER stress in a K205R dose-dependent manner, as indicated by enhanced expression of ATF6 and phosphorylation of PERK, as well as the downstream effectors of ER stress, such as Bip, phosphorylated eIF2α, ATF4, and XBP1 ( Figure 3A). This result suggested that K205R activated the IRE1, PERK, and ATF6 signaling pathways. We also obtained similar results in HeLa cells ( Figure 3B). To further confirm the role of K205R in triggering ER stress, we performed qRT-PCR analysis to examine the transcript level of ER stress-responsive genes. The mRNA levels of ERdj4, processed Xbp1 mRNA We then examined the subcellular distribution of K205R through co-localization assays with markers of lysosomes (indicated by LAMP1), mitochondria (indicated by TOM20), Golgi bodies (indicated by GM130), and ER (indicated by calnexin). K205R exhibited no clear co-localization with LAMP1, TOM20, and GM130, thus suggesting that K205R scarcely localized to lysosomes, mitochondria, and Golgi bodies ( Figure 2C). K205R was diffusely distributed in the cells in a degree similar to calnexin ( Figure 2C). Notably, K205R was found in punctate structures, in addition to areas with diffuse distribution ( Figure 2C). Protein punctate structures may form as protein aggregates or stress granules (SGs) [21,22]. Protein aggregates interact with the selective autophagy receptor P62 and subsequently undergo aggrephagy [21]. We did not observe co-localization of K205R with P62, indicating that K205R did not form protein aggregates ( Figure 2D). Interestingly, K205R clearly colocalized with the SG marker protein TIA-1 ( Figure 2D). We further performed cell fractionation analysis to identify the localization of K205R in the cytosolic and membrane fractions. The low-density lipoprotein receptor is a cell surface receptor that is recycled in the cytosol after ligand binding [23]. K205R was distributed in both the cytosolic and membrane fractions, similar to the low-density lipoprotein receptor ( Figure 2E). In addition, K205R was present in soluble and insoluble forms in the cells ( Figure 2F).

ASFV K205R Activates ER Stress
Cellular stress, such as ER stress, is a strong inducer of SG formation [24]. Therefore, we sought to determine whether K205R induces ER stress. There are three ER stress sensor pathways, IRE1, PERK and ATF6, which are critical to maintain ER homeostasis [25]. Using immunoblotting analysis, we detected that the expression of K205R in 3D4/21 cells stimulated ER stress in a K205R dose-dependent manner, as indicated by enhanced expression of ATF6 and phosphorylation of PERK, as well as the downstream effectors of ER stress, such as Bip, phosphorylated eIF2α, ATF4, and XBP1 ( Figure 3A). This result suggested that K205R activated the IRE1, PERK, and ATF6 signaling pathways. We also obtained similar results in HeLa cells ( Figure 3B). To further confirm the role of K205R in triggering ER stress, we performed qRT-PCR analysis to examine the transcript level of ER stress-responsive genes. The mRNA levels of ERdj4, processed Xbp1 mRNA [Xbp1(s)/Xbp1(t)], Atf4, Gadd34, and Chop were all up-regulated in response to K205R expression ( Figure 3C-G). Treatment of cells with a PERK inhibitor (GSK2606414, GSK) abolished K205R-induced phosphorylation of PERK and eIF2α ( Figure 3H). Together, these results demonstrated that K205R induced ER stress.  Figure 3H). Together, these results demonstrated that K205R induced ER stress.

ASFV K205R Activates Autophagy
We next attempted to examine whether K205R activates autophagy, given that ER stress is a potent trigger of autophagy [26]. We transfected K205R-GFP plasmids into the HeLa cells and then detected LC3 with immunofluorescence analysis. In the control cells, LC3 was spread throughout the cytosol and nucleus ( Figure 4A). In the K205R-expressing cells, LC3 formed punctate structures, a characteristic of autophagosamal membrane formation ( Figure 4A and 4B). We also examined K205R-induced autophagy by immunoblotting analysis. As shown in Figure 4C, increased expression of K205R resulted in enhanced expression levels of L3-II, ATG5, ATG12, and Beclin-1. When

ASFV K205R Activates Autophagy
We next attempted to examine whether K205R activates autophagy, given that ER stress is a potent trigger of autophagy [26]. We transfected K205R-GFP plasmids into the HeLa cells and then detected LC3 with immunofluorescence analysis. In the control cells, LC3 was spread throughout the cytosol and nucleus ( Figure 4A). In the K205R-expressing cells, LC3 formed punctate structures, a characteristic of autophagosamal membrane formation ( Figure 4A,B). We also examined K205R-induced autophagy by immunoblotting analysis. As shown in Figure 4C, increased expression of K205R resulted in enhanced expression levels of L3-II, ATG5, ATG12, and Beclin-1. When autophagy is activated, the selective autophagy receptor P62 is degraded in lysosomes and serves as an indicator of autophagic flux [21]. We observed that P62 expression decreased in response to K205R expression ( Figure 4C). Bafilomycin A1, an inhibitor of the fusion of autophagosomes and lysosomes, can be used to analyze autophagic flux [27]. In the K205R expressing cells, bafilomycin A1 induced more LC3 accumulation than that in the control cells, but caused no P62 degradation ( Figure 4D). Given that ATG5 and Beclin-1 are essential for the formation of autophagosomes [28], we verified the role of K205R in autophagy induction in ATG5 −/− and Beclin-1 −/− cells. K205R failed to induce autophagy in the ATG5 −/− and Beclin-1 −/− cells, as indicated by immunoblotting analysis of LC3-II, ATG5, ATG12, and Beclin-1 ( Figure 4E). Inhibition of PERK by GSK in the K205R-transfected cells resulted in lower expression of LC3-II, ATG5, ATG12, and Beclin-1 than that in the K205R-transfected cells, further suggesting that K205R activated autophagy through ER stress ( Figure 4F). The AKT/mTOR pathway negatively regulates unc-51 like autophagy activating kinase 1 (ULK1), and hence au- Given that ATG5 and Beclin-1 are essential for the formation of autophagosomes [28], we verified the role of K205R in autophagy induction in ATG5 −/− and Beclin-1 −/− cells. K205R failed to induce autophagy in the ATG5 −/− and Beclin-1 −/− cells, as indicated by immunoblotting analysis of LC3-II, ATG5, ATG12, and Beclin-1 ( Figure 4E). Inhibition of PERK by GSK in the K205R-transfected cells resulted in lower expression of LC3-II, ATG5, ATG12, and Beclin-1 than that in the K205R-transfected cells, further suggesting that K205R activated autophagy through ER stress ( Figure 4F). The AKT/mTOR pathway negatively regulates unc-51 like autophagy activating kinase 1 (ULK1), and hence autophagy [29]. Therefore, we attempted to determine whether the AKT/mTOR/ULK1 signaling pathway is involved in K205R-induced autophagy. We observed that phosphorylated AKT and mTOR decreased when cells expressed K205R ( Figure 4G). This result indicated that the AKT/mTOR pathway was inhibited by K205R. Phosphorylation of ULK1 Ser555 was enhanced, whereas phosphorylation of ULK1 Ser757 was decreased in response to K205R expression, suggesting that K205R activated ULK1 ( Figure 4G). Collectively, these data indicated that K205R activated autophagy through the AKT/mTOR/ULK1 signaling pathway.

ASFV K205R Activates the NF-κB Signaling Pathway
It is known that ER stress can elicit proinflammation [25], therefore we sought to determine whether K205R activates the NF-κB signaling pathway. Phosphorylation of the IκBα and P65 subunits of nuclear factor kappa B (NF-κB) is essential for P65 translocation into the nucleus and subsequent NF-κB activation [30]. We observed that K205R expression resulted in the phosphorylation of IκBα and P65, as indicated by immunoblotting analysis ( Figure 5A). Immunofluorescence analysis suggested that P65 was translocated into the nucleus in the presence of K205R expression ( Figure 5B). We further analyzed NF-κB activation by cell fractionation analysis of P65 with β-actin as a cytosolic marker and Lamin B1 as a nuclear marker. As shown in Figure 5C, LPS (a well-known NF-κB activator) stimulated P65's phosphorylation and translocation into the nucleus ( Figure 5C). Expression of K205R-HA with simultaneous treatment of cells with LPS (K205R-HA + LPS) promoted the translocation of phosphorylated P65 into the nucleus ( Figure 5C).
Given that NF-κB activation triggers the expression of proinflammatory cytokines, we next examined the transcription of Il-6, Il-18, and Tnfa by qRT-PCR analysis. K205R increased the mRNA levels of Il-6, Il-18, and Tnfa in a K205R dose-dependent manner ( Figure 5D). K205R stimulated IL-18 secretion into the culture medium, as indicated by ELISA analysis ( Figure 5E). We further verified K205R-induced NF-κB activation in 3D4/21 P65 −/− cells. Neither LPS nor K205R stimulated the transcription of Il-6 and Il-18 in the 3D4/21 P65 −/− cells ( Figure 5F,G). We finally determined whether inhibition of ER stress by a PERK inhibitor might abrogate K205R-mediated activation of the NF-κB signaling pathway. In the K205R expressing cells, GSK treatment prevented the phosphorylation of IκBα and P65 ( Figure 5H). GSK treatment also inhibited the transcription of Il-6, Il-18, and Tnfa, as well as the secretion of IL-18 when K205R-HA was expressed in cells ( Figure 5I,J). Together, these results demonstrated that K205R activated the NF-κB signaling pathway.