Skip to main content
Download PDF
  • Research article
  • Open access
  • Published:

Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation

Veterinary Research volume 55, Article number: 151 (2024) Cite this article

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) has led to significant economic losses in the global swine industry. Type I interferon (IFN-I) plays a crucial role in the host’s resistance to PRRSV infection. Despite extensive research showing that PRRSV employs multiple strategies to antagonise IFN-I induction, the underlying mechanisms remain to be fully elucidated. In this study, we have discovered that PRRSV inhibits the production of IFN-I by degrading TANK-binding kinase 1 (TBK1) through chaperon-mediated autophagy (CMA). From a mechanistic standpoint, PRRSV nonstructural protein 2 (Nsp2) increases the interaction between the heat shock protein member 8 (HSPA8) and TBK1. This interaction leads to the translocation of TBK1 into lysosomes for degradation, mediated by lysosomal-associated membrane protein 2A (LAMP2A). As a result, the downstream activation of IFN regulatory factor 3 (IRF3) and the production of IFN-I are hindered. Together, these results reveal a new mechanism by which PRRSV suppresses host innate immunity and contribute to the development of new antiviral strategies against the virus.

Introduction

Innate immunity is the host’s first line of defence against RNA viruses [1, 2]. It recognises viral RNAs through pattern recognition receptors and activates IFN regulatory factor 3 (IRF3) or the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated type I interferon (IFN-I) production [3,4,5,6]. The expression of IFN-I is crucial in limiting the early replication and spread of viruses [7]. RNA viruses have developed different mechanisms to suppress innate immunity and establish infection [8].

Porcine reproductive and respiratory syndrome (PRRS) is characterised by severe reproductive failure in sows and respiratory diseases in pigs of all ages. This disease has imposed a significant economic burden on the global swine industry [9, 10]. Its causative agent, PRRS virus (PRRSV), is a member of the family Arteriviridae in the order Nidovirales. It is an enveloped, single-stranded positive-sense RNA virus containing a genome of ~15 kb [11, 12]. Two-thirds of the PRRSV genome encodes nonstructural proteins (Nsps), including Nsp1α, Nsp1β, and Nsp2-12, which participate in viral pathogenesis [13,14,15].

Nsps have been shown to antagonise the host’s innate immune responses and facilitate PRRSV persistent infection in vivo (e.g., porcine alveolar macrophages [PAMs]) and in vitro (such as African green monkey kidney cell lines MA-104 and MARC-145) [16,17,18,19]. PRRSV Nsp1α, Nsp4, and Nsp11 all block NF-κB nuclear translocation or eliminate the linear ubiquitination of NF-κB essential modulators, thereby impairing signalling activation and IFN-I production [20,21,22,23,24]. Nsp1β and Nsp2 inhibit the phosphorylation and nuclear translocation of IRF3, which hinders IFN-I expression [25, 26]. Despite these, few studies have previously investigated upstream regulators of IRF3, such as TANK-binding kinase 1 (TBK1).

In this study, we discovered that PRRSV infection leads to the degradation of TBK1. We then identified PRRSV Nsp2 as the factor responsible for TBK1 degradation and outlined the detailed mechanisms involved. Additionally, we found that the degradation of TBK1 by Nsp2 suppresses IRF3-mediated IFN-I production, which in turn facilitates the proliferation of PRRSV.

Materials and methods

Cells and viruses

MARC-145, human embryonic kidney 293 T (HEK-293 T), CRL-2843-CD163, and human cervix carcinoma HeLa cells were stored in our laboratory [27]. CRL-2843-CD163 cells were maintained in Roswell Park Memorial Institute 1640 medium (Cat. No. 31800; Solarbio), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cat. No. 10270–106; Gibco) and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin; Cat. No. P1400; Solarbio) in a humidified 37 Â°C and 5% CO2 incubator. MARC-145 and HEK-293 T cells were maintained in Dulbecco-modified Eagle medium (Cat. No. 12100; Solarbio), supplemented with 10% heat-inactivated FBS and antibiotics. HeLa cells were routinely maintained in modified Eagle medium (Cat. No. 138–0012; iCell Bioscience Inc), supplemented with 10% heat-inactivated FBS and antibiotics at 37 Â°C in 5% CO2.

PRRSV strains HN07-1 (GenBank KX766378.1) and HNhx (GenBank KX766379) were previously isolated by our laboratory [28, 29]. PRRSV strain BJ-4 (GenBank AF331831) was kindly provided by Professor Hanchun Yang of China Agricultural University. PRRSV strain HN07-1 was utilised in this study unless otherwise stated.

Antibodies

Rabbit anti-TBK1 monoclonal antibody (mAb; Cat. No. 3504S), rabbit anti-phospho-TBK1 (p-TBK1) mAb (Cat. No. 5483S), rabbit anti-IRF3 mAb (Cat. No. 11904S), rabbit anti-phospho-IRF3 (p-IRF3) mAb (Cat. No. 79945S), rabbit anti-Flag mAb (Cat. No. 14793), mouse anti-myc mAb (Cat. No. 2276S), and rabbit anti-hemagglutinin (HA) mAb (Cat. No. 3724) were all purchased from Cell Signaling Technology. Rabbit anti-heat shock protein member 8 (HSPA8) polyclonal antibodies (pAbs; Cat. No. 10654–1-AP), mouse anti-lysosomal-associated membrane protein 2A (LAMP2A) mAb (Cat. No. 66301–1-Ig), rabbit anti-β-actin mAb (Cat. No. 81115–1-RR), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (Cat. No. 60004–1-Ig) were purchased from Proteintech. Alexa Fluor 555-goat anti-rabbit IgG pAbs (Cat. No. ab150078), Alexa Fluor 488-goat anti-rabbit IgG pAbs (Cat. No. ab150077), Alexa Fluor 647-goat anti-rabbit IgG pAbs (Cat. No. ab150115), horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG pAbs (Cat. No. ab6721), and HRP-labeled goat anti-mouse IgG pAbs (Cat. No. ab6789) were purchased from Abcam. Rabbit anti-PRRSV nucleocapsid (N) protein pAbs (Cat. No. GTX129270) for immunoblotting (IB) was purchased from GeneTex.

Reagents

Lipofectamine 2000 (Cat. No. 2066194), Lipofectamine LTX with Plus reagent (Cat. No. 15338030), and Lipofectamine RNAiMAX transfection reagent (Cat. No. 13778150) were purchased from Invitrogen. TransIntro® PL Transfection Reagent (Cat. No. FT301-01) was purchased from TransGen Biotech. DMSO (Cat. No. 276855), 3-methyladenine (3-MA; Cat. No. M9281), and MG132 (Cat. No. M7449) were purchased from Sigma-Aldrich. Radioimmunoprecipitation assay (RIPA) lysis buffer (Cat. No. P0013B), NP-40 lysis buffer (Cat. No. P0013F), Western blot (WB) /immunoprecipitation (IP) lysis buffer (Cat. No. P0013), fast silver stain kit (Cat. No. P0017S), Triton X-100 (Cat. No. P0096), and 4’,6-diamidino-2-phenylindole (DAPI; Cat. No. C1006) were all purchased from Beyotime Biotechnology. 0.25% trypsin–EDTA solution (Cat. No. T1320), phosphate buffered solution (PBS; Cat. No. P1010), and 4% paraformaldehyde (PFA; Cat. No. P1110) were purchased from Solarbio. A complete EDTA-free protease inhibitor cocktail (Cat. No. 04693116001) and universal SYBR green Master (Cat. No. 04913914001) were purchased from Roche. 2 × loading buffer (Cat. No. 9173), PrimeScript RT master mix (Cat. No. RR036B), and RNAiso Plus (Cat. No. 9109) were purchased from TaKaRa. Enhanced chemiluminescence (ECL) reagent (Cat. No. P0013B) was purchased from NCM Biotechnology. Chloroquine (CQ; Cat. No. HY-17589A), HA beads (Cat. No. HY-K0201), myc beads (Cat. No. HY-K0206-5), and Flag beads (Cat. No. HY-K0207-5) were purchased from MedChemExpress. PolyI:C (Cat. No. P9582) and 0.22 Âµm polyvinylidene fluoride membranes (Cat. No. ISEQ00010) were obtained from Sigma-Aldrich. The cell viability assay was performed according to our previous study (data not shown) [30].

Plasmid constructs and transfection

The gene of each Nsp or Nsp2-mutant from PRRSV strain HN07-1 was synthesised and cloned into the pCAGGS-HA plasmid. Nsp2 from PRRSV strains BJ-4 and HNhx was cloned into the pCAGGS-HA plasmid. The cDNA of TBK1 was constructed into pCMV-3 × Flag plasmid by GENEWIZ. The full-length HSPA8 was constructed into the pcDNA3.1-myc/his_A and pEGFP-C1 plasmid by our laboratory [31].

MARC-145, HeLa, and HEK-293 T cells were seeded in cell culture plates and grown to 60~70% confluence for the transfection. Following the manufacturers' instructions, the cells were then transfected with the plasmids using Lipofectamine 2000 or TransIntro PL Transfection Reagent. Unless otherwise specified, 12-well cell culture plates were transfected with 1.5 Âµg of each plasmid per well, and six-well cell culture plates were transfected with 2.5 Âµg of each plasmid per well.

Quantitative real-time PCR (RT-qPCR)

Total RNAs were extracted using TRIzol reagent (Cat. No.15596018; Invitrogen), and then reversely transcribed into cDNA using a PrimeScript RT reagent kit (Cat. No. RR037A) in accordance with the manufacturer’s instructions. RT-qPCR amplified the cDNAs from different samples to quantify N abundance using GAPDH mRNA as an endogenous control. The RT-qPCR was performed using a universal SYBR green master on LightCycler480 II (Roche, Basel, Switzerland). The fold change was calculated using the 2−△△CT method [32]. The primers are listed in Table 1.

Table 1 The primers used for RT-qPCR in this study
Full size table

Immunoblotting (IB)

The cells were harvested and then lysed on ice using WB/IP lysis buffer containing a protease inhibitor cocktail. Whole-cell lysates were normalised to equal amounts of GAPDH/β-actin, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto 0.22 Âµm polyvinylidene fluoride membranes. The membranes were blocked in 5% skimmed milk at room temperature (RT) for 2 h and then incubated with the primary antibodies at 4 Â°C overnight. After incubation with the corresponding HRP-conjugated secondary antibodies at RT for 1 h, the IB results were visualised using ECL reagent. Representative images were provided.

Co-immunoprecipitation (co-IP)

The indicated plasmids were expressed in HEK-293 T cells for 36 h. The cells were lysed with WB/IP lysis buffer containing a protease inhibitor cocktail at 4 Â°C for 30 min and centrifuged at 12 000 × g for 10 min. The supernatant was incubated overnight with anti-HA magnetic beads, anti-Flag magnetic beads, or anti-myc magnetic beads at 4 Â°C. The precipitated immune complexes were collected with a magnetic holder, washed with cold PBS, eluted with a 2 × loading buffer, and subjected to IB with the indicated antibodies.

Confocal microscopy

MARC-145 and HeLa cells were transfected with the indicated plasmids or infected with PRRSV for indicated periods. The cells were washed with PBS, fixed with 4% PFA at RT for 10 min, and permeabilised with 0.1% Triton X-100 in PBS at RT for 10 min. After three rinses with cold PBS, the cells were blocked with 5% bovine serum albumin at RT for 1.5 h. After blocking, the cells were incubated overnight with the primary antibodies at 4 Â°C. After three washes with PBS, the cells were incubated with the appropriate fluorescent secondary antibodies at RT for 1 h. The cell nuclei were stained with DAPI for an additional 5 min. All images were taken and processed using a fluorescence microscope (LSM800, Carl Zeiss AG, Oberkochen, Germany) with a confocal laser scanning setup (20 × , 40 × , or 63 ×). The images represent a single slice from a stack obtained from three independent experiments [33]. The co-localisation analyses were conducted using the JaCoP plugin in ImageJ software following established research guidelines [34,35,36]. Pearson’s correlation coefficient (> 0.5) describes the correlation of the intensity distribution between channels. Manders’ correlation coefficient (> 0.6) indicates the actual overlap of the signals, representing the true degree of colocalisation [36, 37].

HA-IP/mass spectrometry (MS)

HEK-293 T cells were transfected with the plasmid expressing Nsp2-HA or HA-tagged empty vector using Lipofectamine 2000 for 36 h, and then were lysed on ice with WB/IP lysis buffer for 30 min. The cell lysates were centrifuged at 12 000 × g for 5 min. Anti-HA magnetic beads were incubated overnight with the supernatants at 4 Â°C. The beads were washed six times with PBS. The associated proteins were analysed by 12.5% SDS-PAGE, and the protein bands in the gel were stained with a fast silver stain kit. The protein bands were excised and subjected to liquid chromatography and tandem MS (LC–MS/MS) by Lumingbio (Shanghai, China). The top-ranked peptide matches were considered for protein identification, and representative images were displayed.

RNA interference

HEK-293 T cells were transfected with small interference RNA (siRNA) targeting HSPA8 (siHSPA8) or LAMP2A (siLAMP2A) using Lipofectamine RNAiMAX according to the manufacturer’s instructions at a final concentration of 50 nM for 36 h. For HSPA8 knockdown during PRRSV infection, MARC-145 or CRL-2843-CD163 cells were infected with PRRSV at a multiplicity of infection (MOI) of 1 for 2 h, and then transfected with siHSPA8 or siRNA-negative control (siNC) for 48 h. For LAMP2A knockdown, MARC-145 or CRL-2843-CD163 cells were transfected with siLAMP2A or siNC. At 24 h post-transfection, the cells were infected with PRRSV at an MOI of 1. RT-qPCR or IB detected the knockdown efficiencies. The siRNAs were designed and synthesised by GenePharma (Shanghai, China). The cells were then used for subsequent experiments. The siRNA sequences are listed in Table 2. The cell viability assay was performed according to our previous study (data not shown) [30].

Table 2 The siRNAs used in this study
Full size table

Flow cytometry (FCM)

The MARC-145 cells infected with PRRSV were treated with 0.25% trypsin–EDTA solution, centrifuged at 2000 × g, and re-suspended in PBS. The cells were fixed with 4% PFA, permeabilised with 0.1% Triton X-100, and then blocked with 5% bovine serum albumin. The cells were incubated with or without mouse anti-N mAb at RT for 2 h, and Alexa Fluor 647-goat anti-mouse IgG pAbs at RT for 1 h. Finally, the washed cells were resuspended in PBS and the percentage of PRRSV-infected cells was analysed by a flow cytometer (CytoFLEX; Beckman Coulter, Brea, USA). Representative images were shown.

PRRSV titration assay

The PRRSV-infected cells were subjected to three freeze–thaw cycles and centrifuged to collect the supernatant. The progeny virus titers were measured by detecting the 50% tissue culture infected dose (TCID50) in MARC-145 cells. In brief, MARC-145 cells were seeded in 96-well cell culture plates and inoculated with a tenfold serial dilution of the supernatant at 37 Â°C for 1 h. The excess inoculum was removed by washing it with PBS. The cells were cultured for 3 to 6 days. Cytopathic effects were observed using an inverted microscope (Axiovert 40, Carl Zeiss AG, Oberkochen, Germany). The TCID50 value was calculated according to the method of Reed-Muench [38].

Statistical analysis

Each experiment included three replicates and was independently repeated at least three times. The data are presented as means ± standard error of the mean (SEM). Prism 8.0 software (San Diego, USA) performed all data and calculations using the unpaired two-tailed Student t test. Statistical significance was represented by asterisks (ns, not significant [P > 0.05]; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Results

PRRSV Nsp2 degrades TBK1

Firstly, we examined the RNA and protein levels of endogenous TBK1 in MARC-145 cells infected with the highly pathogenic (HP)-PRRSV strain HN07-1-infected using RT-qPCR and IB. As shown in Figures 1A and 1B, TBK1 RNA levels remained stable, while protein levels decreased significantly in a dose-dependent manner. We observed decreased TBK1 protein levels at 12, 24, and 36 h after HP-PRRSV infection (hpi, Figure 1C). Furthermore, we confirmed that the low pathogenic PRRSV strain BJ-4 and the NADC30-like PRRSV strain HNhx decreased TBK1 abundance (Figure 1D). All these results show that PRRSV infection degrades TBK1.

Figure 1
figure 1

PRRSV Nsp2 degrades TBK1. A, B MARC-145 cells were infected with PRRSV strain HN07-1 at different MOIs (0.5, 1, and 5 MOI) or mock-infected and collected at 24 hpi. A The relative TBK1 mRNA abundance was analysed using RT-qPCR. Statistical analysis was carried out using the Student t test. ns, not significant (P > 0.05) B The TBK1 protein abundance was detected using IB. C MARC-145 cells were infected with PRRSV strain HN07-1 at an MOI of 1 or mock infected for indicated time periods (12, 24, or 36 h). The TBK1 protein level was detected by IB. D The mock-infected, or PRRSV strain HN07-1, BJ-4, or HNhx-infected (MOI = 1) MARC-145 cells were collected at 24 hpi. The TBK1 protein level was detected by IB. E HEK-293 T cells were transfected with the plasmids encoding each PRRSV strain HN07-1 Nsp-HA and Flag-TBK1 or HA-tagged empty vector, followed by IB analyses with the specific antibodies. Asterisks mark the target proteins. F The Nsp2-HA plasmid (0.5, 1, or 1.5 Âµg) was co-transfected with the Flag-TBK1 plasmid into HEK-293 T cells for 24 h. IB detected the TBK1 protein level. G HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA of PRRSV strain HN07-1, BJ-4, or HNhx and Flag-TBK1 or HA-tagged empty vector, followed by IB analyses with the specific antibodies.

Full size image

To identify which PRRSV Nsps degraded TBK1, we overexpressed each HA-tagged HP-PRRSV strain HN07-1 Nsp (Nsp-HA) and Flag-tagged-TBK1 (Flag-TBK1) in the HEK-293 T cells, and detected their expression and TBK1 protein levels. As shown in Figure 1E, Nsp1α-HA, Nsp1β-HA, Nsp2-HA, Nsp4-HA, Nsp5-HA, Nsp7-HA, and Nsp9-12-HA were successfully expressed, and among them, Nsp1β-HA, Nsp2-HA, Nsp5-HA, and Nsp9-11-HA degraded TBK1. As our laboratory has been investigating Nsp2 [39], we have proceeded with further research focusing on Nsp2-induced degradation of TBK1. We observed that the overexpression of HP-PRRSV Nsp2-HA led to a reduction in the abundance of exogenous TBK1 in a manner that depended on the dosage (Figure 1F). We verified that the Nsp2-HA of PRRSV strains BJ-4 and HNhx reduced TBK1 protein levels (Figure 1G). These data demonstrate that PRRSV Nsp2 degrades TBK1.

PRRSV Nsp2 interacts with TBK1 and induces its degradation via lysosomal pathway

We next examined whether PRRSV Nsp2 interacted with TBK1. We co-transfected the Nsp2-HA and Flag-TBK1 plasmids into HEK-293 T cells and performed IP using anti-HA/Flag magnetic beads. As shown in Figure 2A, we detected an interaction between exogenous Nsp2-HA and Flag-TBK1. Meanwhile, we overexpressed Nsp2-HA and Flag-TBK1 in HeLa cells and observed their co-localisation via confocal microscopy. Their co-localisation was further quantified using Pearson’s correlation coefficient, and the mean value was 0.745, indicating an interaction between these two proteins (Figure 2B). In addition, we monitored that endogenous Nsp2 interacted with TBK1 by IP and confocal microscopy in the PRRSV-infected MARC-145 cells (Figures 2C, D). These results substantiate that PRRSV Nsp2 specifically interacts with TBK1.

Figure 2
figure 2

PRRSV Nsp2 interacts with TBK1 and induces its degradation via the lysosomal pathway. A HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA and Flag-TBK1 or HA/Flag-tagged empty vector for 36 h, followed by co-IP with anti-HA/Flag magnetic beads and IB analyses with anti-HA/Flag antibodies. B HeLa cells were transfected with the plasmids encoding Nsp2-HA and Flag-TBK1 for 24 h. In parallel, HeLa cells were transfected with the plasmid encoding Nsp2-HA and Flag-tagged empty vector, or Flag-TBK1 and HA-tagged empty vector. Flag-TBK1 and Nsp2-HA were visualised with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. The fluorescent signals were observed with confocal microscopy (scale bars = 10 Âµm). The co-localisation was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. C MARC-145 cells were infected with PRRSV at an MOI of 1 for 24 h. They were then analysed via endogenous IP using protein A/G magnetic beads pre-incubated with anti-Nsp2 pAbs, and IB with anti-Nsp2 and anti-TBK1 antibodies. D MARC-145 cells were infected with PRRSV at an MOI of 0.1 for 24 h. The fluorescent signals were observed with confocal microscopy (scale bars = 10 Âµm). The co-localisation was assessed by determining the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. E HEK-293 T cells were co-transfected with the plasmids encoding Flag-TBK1 and Nsp2-HA or Nsp2-mutant-HA. At 24 h post-transfection, the cell lysates were collected to analyse the TBK1 protein level with IB. F HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA and Flag-TBK1 for 6 h. 3-MA, CQ, MG132, or DMSO was added and the samples were collected after 24 h for IB analyses with anti-HA and anti-Flag antibodies.

Full size image

Considering their interaction, we initially assessed whether Nsp2 directly degrades TBK1. The PRRSV Nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities, with highly conserved C55, C111, H124, C142, and C147 being responsible for the activities [40]. We mutated these residues to alanine (named Nsp2-mutant-HA) and detected that Nsp2-mutant-HA still degraded TBK1 (Figure 2E), indicating that its degradation was independent of Nsp2 cysteine protease activity. As the autolysosomal pathway and ubiquitin–proteasome system are two main host cellular degradation routes [41, 42], we investigated whether PRRSV Nsp2 degrades TBK1 through these two routes using the autophagy inhibitor 3-MA, lysosomal inhibitor CQ, and the proteasome inhibitor MG132 [43,44,45]. As shown in Figure 2F, only CQ treatment reversed TBK1 degradation, suggesting that Nsp2 induces TBK1 degradation in a lysosome-dependent manner.

PRRSV Nsp2 interacts with HSPA8

To understand how Nsp2 breaks down TBK1 using lysosomes, we screened the host cellular proteins targeted by PRRSV Nsp2. We used IP along with LC–MS/MS. In cells overexpressing Nsp2-HA, we observed a distinct protein band marked by a red arrow (~71 kDa), identified as HSPA8 (Figure 3A).

Figure 3
figure 3

PRRSV Nsp2 interacts with HSPA8. A HEK-293 T cells were transfected with the plasmid expressing HA or Nsp2-HA. The proteins were immunoprecipitated in cell lysates using an anti-HA antibody, separated by 12% SDS-PAGE, and stained with silver. The red arrow indicates the significantly different immunoprecipitated protein band. The black arrow marks Nsp2-HA. The panel on the right shows the tandem MS analysis of HSPA8 peptides. B HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA and HSPA8-myc or HA/myc-tagged empty vector for 36 h, followed by co-IP with anti-HA or anti-myc magnetic beads, and IB analyses with anti-HA and anti-myc antibodies. C HeLa cells were transfected with the plasmids encoding Nsp2-HA and HSPA8-myc for 24 h. In parallel, HeLa cells were transfected with the plasmid encoding Nsp2-HA and myc-tagged empty vector, or HSPA8-myc and HA-tagged empty vector. HSPA8-myc and Nsp2-HA were visualised with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. The fluorescent signals were observed with confocal microscopy (scale bars = 10 Âµm). The co-localisation was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. D MARC-145 cells were infected with PRRSV at an MOI of 1 for 24 h. They were then analysed via endogenous IP using protein A/G magnetic beads pre-incubated with anti-Nsp2 pAbs, and IB with anti-Nsp2 and anti-HSPA8 antibodies. E MARC-145 cells were infected with PRRSV at an MOI of 1 for 24 h. The fluorescent signals were observed with confocal microscopy (scale bars = 10 Âµm). The co-localisation was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software.

Full size image

To confirm that Nsp2 interacted with HSPA8, we co-transfected the Nsp2-HA and HSPA8-myc plasmids in HEK-293 T cells and detected an interaction between exogenous Nsp2 and HSPA8 (Figure 3B). Furthermore, we overexpressed Nsp2-HA and HSPA8-myc in HeLa cells and observed their co-localisation via confocal microscopy (Figure 3C). Moreover, we conducted IP and confocal microscopy in the PRRSV-infected cells and monitored endogenous Nsp2 interacting with HSPA8 (Figures 3D, E). These data indicate that PRRSV Nsp2 specifically interacts with HSPA8.

PRRSV Nsp2 degrades TBK1 via chaperone-mediated autophagy (CMA)

HSPA8 recognises the Lys-Phe-Glu-Arg-Gln (KFERQ) motif on target proteins and forms HSPA8-substrate complexes; the complexes are subsequently translocated into lysosomes via LAMP2A for degradation, namely CMA [46]. It has been reported that HSPA8 interacts with TBK1 and degrades it via CMA [47]. Therefore, we hypothesised that Nsp2 enhances the interaction between HSPA8 and TBK1 to promote its degradation. We detected that Nsp2-HA overexpression strengthened the interaction of HSPA8-myc with Flag-TBK1 (Figures 4A, B). We also found that TBK1 localisation in lysosomes increased in PRRSV-infected MARC-145 cells (the Manders’ correlation coefficient increased from 0.617 to 0.889, Figure 4C). We then applied siHSPA8 or siLAMP2A and found that the knockdown of HSPA8 and LAMP2A reversed TBK1 degradation by Nsp2-HA (Figure 4D). Similarly, TBK1 degradation was antagonised during PRRSV infection in the HSPA8 and LAMP2A knockdown cells (Figure 4E). Taken together, these data provide evidence that Nsp2 degrades TBK1 via CMA.

Figure 4
figure 4

PRRSV Nsp2 degrades TBK1 via CMA. A HEK-293 T cells were transfected with the plasmids encoding Flag-TBK1, HSPA8-myc, and Nsp2-HA or HA-tagged empty vector for 36 h. Co-IP was performed with anti-myc magnetic beads and IB was conducted with the specific antibodies. B HeLa cells were transfected with the plasmids encoding Flag-TBK1, HSPA8-myc, and Nsp2-HA or HA-tagged empty vector for 36 h. The fluorescent signals were observed with confocal microscopy. The co-localisation was assessed by determination of the Pearson’s correlation coefficient (scale bars = 10 Âµm) using the JaCoP plugin in ImageJ software. C MARC-145 cells were infected with PRRSV at 0.1 MOI for 24 h. The fluorescent signals were observed with confocal microscopy. The co-localisation was assessed by determination of the Manders’ correlation coefficient (scale bars = 10 Âµm) using the JaCoP plugin in ImageJ software. D HEK-293 T cells were transfected with siHSPA8, siLAMP2A, or siNC, and the plasmids encoding Flag-TBK1 and Nsp2-HA. The samples were collected after 36 h for IB analyses with the specific antibodies. E MARC-145 cells were transfected with siHSPA8/LAMP2A or siNC to detect their effects during PRRSV infection, and IB was conducted with the specific antibodies. The data are presented as means ± SEM from three independent experiments. Statistical analysis was carried out using the Student t test. **, P < 0.01, ****, P < 0.0001.

Full size image

PRRSV Nsp2 degrades TBK1 via CMA to suppress IRF3 activation and IFN production

TBK1 activates IRF3 signalling and initiates IFN production [48]. Consequently, we speculated that TBK1 degradation by Nsp2 via CMA hampered the p-TBK1 level, thereby hindering p-IRF3 levels and IFN production. As expected, overexpression of Nsp2-HA decreased the TBK1, p-TBK1, and p-IRF3 levels (Figure 5A). In contrast, the knockdown of HSPA8 and LAMP2A reversed the Nsp2-HA-induced degradation of TBK1 and increased the p-TBK1 and p-IRF3 levels (Figures 5B, C). Additionally, HSPA8 and LAMP2A knockdown enhanced IFN-β production (Figure 5D). These data show that PRRSV Nsp2 degrades TBK1 through CMA to inhibit IRF3 activation and IFN production.

Figure 5
figure 5

PRRSV Nsp2 degrades TBK1 via CMA to suppress IRF3 activation and IFN production. A HEK-293 T cells were transfected with the plasmid encoding Nsp2-HA or HA-tagged empty vector. The PolyI:C was added 12 h before the sample was collected. IB was conducted with the specific antibodies. B, C HEK-293 T cells were transfected with siHSPA8, siLAMP2A, or siNC, and the plasmid encoding Nsp2-HA. The PolyI:C was added 12 h before the sample was collected. IB was conducted with the specific antibodies. D HEK-293 T cells were transfected with siHSPA8, siLAMP2A, or siNC, and the plasmid encoding Nsp2-HA. The PolyI:C was added 12 h before the sample was collected. IFN-β RNA abundance was detected using RT-qPCR. The data are presented as means ± SEM from three independent experiments. Statistical analysis was carried out using the Student t test. **, P < 0.01.

Full size image

PRRSV degrades TBK1 via CMA to suppress innate immunity and promote viral proliferation

We further investigated the impact of PRRSV-induced TBK1 degradation via CMA on the host's innate immunity and viral proliferation. Figures 6A, B show that the knockdown of HSPA8/LAMP2A enhanced the TBK1, p-TBK1, and p-IRF3 levels during PRRSV infection. Meanwhile, HSPA8/LAMP2A knockdown increased IFN-β mRNA abundance while decreasing PRRSV RNA levels, as detected by RT-qPCR (Figures 6C, D). FCM results also showed that intracellular PRRSV infection was lowered (~50% reduction in the HSPA8 knockdown cells and ~80% reduction in the LAMP2A knockdown ones, Figure 6E). As PAMs are primary in vivo target cells for PRRSV infection, we further performed the assays in a continuous PAM cell line CRL-2843-CD163, which stably expresses PRRSV indispensable receptor CD163 [49, 50]. Similarly, siHSPA8/siLAMP2A increased IFN-β RNA abundance and decreased PRRSV RNA levels (Figure 6F). The knockdown of HSPA8 also reduced PRRSV titres by at least tenfold (> 1 log10TCID50 mL−1, Figure 6G). These results show that PRRSV degrades TBK1 through CMA to suppress innate immunity and facilitate viral proliferation.

Figure 6
figure 6

PRRSV degrades TBK1 via CMA to suppress innate immunity and facilitate viral proliferation. A MARC-145 cells were infected with PRRSV at an MOI of 1 for 2 h, then transfected with siHSPA8 or siNC for 48 h, and IB was conducted with the specific antibodies. B MARC-145 cells were transfected with siLAMP2A or siNC for 24 h. At 24 h post-transfection, the cells were infected with PRRSV at an MOI of 1 and IB was conducted with the specific antibodies. C–E MARC-145 cells were transfected with siHSPA8/siLAMP2A or siNC to detect their effects during PRRSV infection. C IFN-β RNA abundance was detected using RT-qPCR. D PRRSV RNA abundance was detected using RT-qPCR. E PRRSV infection was detected using FCM. F CRL-2843-CD163 cells were transfected with siHSPA8/siLAMP2A and the cell lysates were collected to detect their effects during PRRSV infection. IFN-β RNA and PRRSV RNA abundance were detected using RT-qPCR. G CRL-2843-CD163 cells were transfected with siHSPA8 or siNC. PRRSV titers were measured by assessing TCID50. The data are presented as means ± SEM from three independent experiments. Statistical analysis was carried out using the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0011; ****, P < 0.0001.

Full size image

Discussion

PRRSV employs various strategies to suppress innate immunity and establish persistent infection, posing a major obstacle to PRRS eradication [14]. Therefore, it is crucial to understand the interaction between PRRSV and the host’s innate immunity to elucidate PRRSV pathogenesis and improve prevention and control strategies. PRRSV Nsps antagonise the host’s innate immune responses [51]. However, there are limited studies on their antagonism of TBK1-mediated IFN production.

TBK1 is a kinase that mediates innate immune signalling. TBK1 phosphorylation leads to downstream IRF3 activation and IFN-I responses [52, 53], and is strictly regulated by several mechanisms [48, 54]. For example, the E3 ubiquitin ligase DTX4 promotes the polyubiquitination of TBK1 at Lys670 with K48 linkage, resulting in TBK1 degradation and the inhibition of immune responses [55].

In this study, we observed that different PRRSV strains degrade TBK1, and we further identified that PRRSV Nsp2 degrades TBK1 (Figure 1). Nsp2 carries out papain-like cysteine protease activities and cleaves viral polyproteins to produce Nsps as well as other target proteins for PRRSV replication [39, 40]. Interestingly, we found that PRRSV Nsp2 interacted with TBK1 but degraded TBK1 independent of its cysteine protease activity (Figure 2). Subsequently, we showed that PRRSV Nsp2 degrades TBK1 via CMA, which reveals a new mechanism for protein degradation by Nsp2.

CMA is a form of selective autophagy, where proteins containing the KFERQ motif are recognised by HSPA8 and then translocated into lysosomes via LAMP2A for degradation [46]. It has been shown to participate in TBK1 degradation [47]. We confirmed that PRRSV Nsp2 interacts with HSPA8 and enhances its interaction with TBK1 to degrade TBK1 via CMA (Figures 3 and 4).

Finally, PRRSV Nsp2-induced TBK1 degradation via CMA decreased TBK1 phosphorylation levels and inhibited downstream IRF3 activation. This suppressed IFN-I production and promoted viral proliferation (Figures 5 and 6). PRRSV Nsp2 is well-known for its involvement in immunosuppression [26, 56,57,58]. In detail, Nsp2 impedes the transport of stimulator of IFN genes from the endoplasmic reticulum to the Golgi apparatus and blocks IFN signalling [56]. In addition, Nsp2 inhibits the signalling of melanoma differentiation-associated gene 5, which impairs immune responses [57]. Nsp2 also antagonises NF-κB-mediated immune responses by deubiquitinating K48-linked I-kappa-B-alpha [58]. Moreover, Nsp2 prevents the phosphorylation and nuclear translocation of IRF3, thereby reducing the expression of IFN-I [26]. Our study reveals a different mechanism through which Nsp2 suppresses the host’s innate immunity.

Based on these results, we propose a model to describe how PRRSV degrades TBK1 via CMA to suppress host innate immunity and facilitate viral proliferation (Figure 7). Our results contribute to a comprehensive understanding of PRRSV immunosuppression and provide promising targets for the prevention and control of PRRS.

Figure 7
figure 7

Schematic model depicting that PRRSV inhibits IFN-I production by degrading TBK1 via CMA, thereby promoting viral proliferation. Mechanistically, PRRSV Nsp2 enhances the interaction between HSPA8 and TBK1, leading to LAMP2A-mediated translocation of TBK1 into lysosomes for degradation, which impedes downstream IRF3 signalling and IFN-I production.

Full size image

Availability of data and materials

The data generated during this study are available from the corresponding authors upon reasonable request.

Abbreviations

PRRSV:

porcine reproductive and respiratory syndrome virus

IFN-I:

type I interferon

TBK1:

TANK-binding kinase 1

CMA:

chaperon-mediated autophagy

Nsp2:

nonstructural protein 2

HSPA8:

heat shock protein member 8

LAMP2A:

lysosomal-associated membrane protein 2A

IRF3:

IFN regulatory factor 3

NF-κB:

nuclear factor kappa-light-chain-enhancer of activated B cells

PRRS:

porcine reproductive and respiratory syndrome

PAMs:

porcine alveolar macrophages

HEK-293 T:

human embryonic kidney 293 T

FBS:

fetal bovine serum

mAb:

monoclonal antibody

p-TBK1:

phospho-TBK1

p-IRF3:

phospho-IRF3

HA:

hemagglutinin

pAbs:

polyclonal antibodies

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

HRP:

horseradish peroxidase

N:

nucleocapsid

IB:

immunoblotting

RIPA:

radioimmunoprecipitation assay

WB/IP:

Western blot/immunoprecipitation

DAPI:

4′,6-Diamidino-2-phenylindole

ECL:

enhanced chemiluminescence

RT-qPCR:

quantitative real-time PCR

SDS-PAGE:

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

RT:

room temperature

MS:

mass spectrometry

LC–MS/MS:

liquid chromatography and tandem MS

siRNA:

small interference RNA

MOI:

multiplicity of infection

siNC:

SiRNA-negative control

FCM:

flow cytometry

TCID50 :

50% Tissue culture infected dose

SEM:

standard error of the mean

hpi:

hours post-infection

References

  1. Müller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M (1994) Functional role of type I and type II interferons in antiviral defense. Science 264:1918–1921

    Article  PubMed  Google Scholar 

  2. Sadler AJ, Williams BR (2008) Interferon-inducible antiviral effectors. Nat Rev Immunol 8:559–568

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. O’Neill LA, Bowie AG (2010) Sensing and signaling in antiviral innate immunity. Curr Biol 20:R328-333

    Article  CAS  PubMed  Google Scholar 

  4. Liu G, Gack MU (2020) Distinct and orchestrated functions of RNA sensors in innate immunity. Immunity 53:26–42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hiscott J (2007) Triggering the innate antiviral response through IRF-3 activation. J Biol Chem 282:15325–15329

    Article  CAS  PubMed  Google Scholar 

  6. Hiscott J, Grandvaux N, Sharma S, Tenoever BR, Servant MJ, Lin R (2003) Convergence of the NF-kappaB and interferon signaling pathways in the regulation of antiviral defense and apoptosis. Ann N Y Acad Sci 1010:237–248

    Article  CAS  PubMed  Google Scholar 

  7. Stetson DB, Medzhitov R (2006) Type I interferons in host defense. Immunity 25:373–381

    Article  CAS  PubMed  Google Scholar 

  8. Chathuranga K, Weerawardhana A, Dodantenna N, Lee JS (2021) Regulation of antiviral innate immune signaling and viral evasion following viral genome sensing. Exp Mol Med 53:1647–1668

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Valdes-Donoso P, Jarvis LS (2022) Combining epidemiology and economics to assess control of a viral endemic animal disease: Porcine Reproductive and Respiratory Syndrome (PRRS). PLoS One 17:e0274382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lunney JK, Benfield DA, Rowland RR (2010) Porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine. Virus Res 154:1–6

    Article  CAS  PubMed  Google Scholar 

  11. Dokland T (2010) The structural biology of PRRSV. Virus Res 154:86–97

    Article  CAS  PubMed  Google Scholar 

  12. Conzelmann KK, Visser N, Van Woensel P, Thiel HJ (1993) Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology 193:329–339

    Article  CAS  PubMed  Google Scholar 

  13. Fang Y, Snijder EJ (2010) The PRRSV replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins. Virus Res 154:61–76

    Article  CAS  PubMed  Google Scholar 

  14. Lunney JK, Fang Y, Ladinig A, Chen N, Li Y, Rowland B, Renukaradhya GJ (2016) Porcine reproductive and respiratory syndrome virus (PRRSV): pathogenesis and interaction with the immune system. Annu Rev Anim Biosci 4:129–154

    Article  CAS  PubMed  Google Scholar 

  15. Music N, Gagnon CA (2010) The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis. Anim Health Res Rev 11:135–163

    Article  PubMed  Google Scholar 

  16. Kim HS, Kwang J, Yoon IJ, Joo HS, Frey ML (1993) Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol 133:477–483

    Article  CAS  PubMed  Google Scholar 

  17. Duan X, Nauwynck HJ, Pensaert MB (1997) Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV). Vet Microbiol 56:9–19

    Article  CAS  PubMed  Google Scholar 

  18. Duan X, Nauwynck HJ, Pensaert MB (1997) Effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV). Arch Virol 142:2483–2497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wills RW, Doster AR, Galeota JA, Sur JH, Osorio FA (2003) Duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus. J Clin Microbiol 41:58–62

    Article  PubMed  PubMed Central  Google Scholar 

  20. Song C, Krell P, Yoo D (2010) Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-β production by porcine reproductive and respiratory syndrome virus. Virology 407:268–280

    Article  CAS  PubMed  Google Scholar 

  21. Huang C, Zhang Q, Guo XK, Yu ZB, Xu AT, Tang J, Feng WH (2014) Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-κB essential modulator. J Virol 88:10934–10945

    Article  PubMed  PubMed Central  Google Scholar 

  22. Chen J, Wang D, Sun Z, Gao L, Zhu X, Guo J, Xu S, Fang L, Li K, Xiao S (2019) Arterivirus nsp4 antagonizes interferon beta production by proteolytically cleaving NEMO at multiple sites. J Virol 93:e00385-19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wei ZY, Liu F, Li Y, Wang HL, Zhang ZD, Chen ZZ, Feng WH (2020) Aspartic acid at residue 185 modulates the capacity of HP-PRRSV nsp4 to antagonize IFN-I expression. Virology 546:79–87

    Article  CAS  PubMed  Google Scholar 

  24. Su Y, Shi P, Zhang L, Lu D, Zhao C, Li R, Zhang L, Huang J (2018) The superimposed deubiquitination effect of OTULIN and porcine reproductive and respiratory syndrome virus (PRRSV) Nsp11 promotes multiplication of PRRSV. J Virol 92:e00175-18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Beura LK, Sarkar SN, Kwon B, Subramaniam S, Jones C, Pattnaik AK, Osorio FA (2010) Porcine reproductive and respiratory syndrome virus nonstructural protein 1beta modulates host innate immune response by antagonizing IRF3 activation. J Virol 84:1574–1584

    Article  CAS  PubMed  Google Scholar 

  26. Li H, Zheng Z, Zhou P, Zhang B, Shi Z, Hu Q, Wang H (2010) The cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein 2 antagonizes interferon regulatory factor 3 activation. J Gen Virol 91:2947–2958

    Article  CAS  PubMed  Google Scholar 

  27. Liu Y, Li R, Chen XX, Zhi Y, Deng R, Zhou EM, Qiao S, Zhang G (2019) Nonmuscle myosin heavy chain IIA recognizes sialic acids on sialylated RNA viruses to suppress proinflammatory responses via the DAP12-syk pathway. MBio 10:e00574-19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Qiao S, Feng L, Bao D, Guo J, Wan B, Xiao Z, Yang S, Zhang G (2011) Porcine reproductive and respiratory syndrome virus and bacterial endotoxin act in synergy to amplify the inflammatory response of infected macrophages. Vet Microbiol 149:213–220

    Article  CAS  PubMed  Google Scholar 

  29. Wang LJ, Wan B, Guo Z, Qiao S, Li R, Xie S, Chen XX, Zhang G (2018) Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in china. Virus Genes 54:86–97

    Article  CAS  PubMed  Google Scholar 

  30. Zheng XX, Li R, Qiao S, Chen XX, Zhang L, Lu Q, Xing G, Zhou EM, Zhang G (2021) Vimentin rearrangement by phosphorylation is beneficial for porcine reproductive and respiratory syndrome virus replication in vitro. Vet Microbiol 259:109133

    Article  CAS  PubMed  Google Scholar 

  31. Wang L, Li R, Geng R, Zhang L, Chen XX, Qiao S, Zhang G (2022) Heat shock protein member 8 (HSPA8) is involved in porcine reproductive and respiratory syndrome virus attachment and internalization. Microbiol Spectr 10:e0186021

    Article  PubMed  Google Scholar 

  32. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108

    Article  CAS  PubMed  Google Scholar 

  33. Wei X, Li R, Qiao S, Chen XX, Xing G, Zhang G (2020) Porcine reproductive and respiratory syndrome virus utilizes viral apoptotic mimicry as an alternative pathway to infect host cells. J Virol 94:e00709-20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zinchuk V, Grossenbacher-Zinchuk O (2009) Recent advances in quantitative colocalization analysis: focus on neuroscience. Prog Histochem Cytochem 44:125–172

    Article  CAS  PubMed  Google Scholar 

  35. Bolte S, Cordelières FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232

    Article  CAS  PubMed  Google Scholar 

  36. Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:C723-742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jensen EC (2013) Quantitative analysis of histological staining and fluorescence using ImageJ. Anat Rec (Hoboken) 296:378–381

    Article  PubMed  Google Scholar 

  38. Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27:493–497

    Article  Google Scholar 

  39. Ss Z, Qian Q, Chen Xx LuQ, Xing G, Qiao S, Li R, Zhang G (2024) Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication. J Virol 98:e0184223

    Article  Google Scholar 

  40. Han J, Rutherford MS, Faaberg KS (2009) The porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities. J Virol 83:9449–9463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741

    Article  CAS  PubMed  Google Scholar 

  42. Wang Y, Le WD (2019) Autophagy and ubiquitin-proteasome system. Adv Exp Med Biol 1206:527–550

    Article  CAS  PubMed  Google Scholar 

  43. Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, Ong CN, Codogno P, Shen HM (2010) Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem 285:10850–10861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Guo N, Peng Z (2013) MG132, a proteasome inhibitor, induces apoptosis in tumor cells. Asia Pac J Clin Oncol 9:6–11

    Article  PubMed  Google Scholar 

  45. Ferreira PMP, Sousa RWR, Ferreira JRO, Militão GCG, Bezerra DP (2021) Chloroquine and hydroxychloroquine in antitumor therapies based on autophagy-related mechanisms. Pharmacol Res 168:105582

    Article  CAS  PubMed  Google Scholar 

  46. Cuervo AM, Wong E (2014) Chaperone-mediated autophagy: roles in disease and aging. Cell Res 24:92–104

    Article  CAS  PubMed  Google Scholar 

  47. Zhao X, Di Q, Yu J, Quan J, Xiao Y, Zhu H, Li H, Ling J, Chen W (2022) USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy. Autophagy 18:891–908

    Article  CAS  PubMed  Google Scholar 

  48. Liu S, Cai X, Wu J, Cong Q, Chen X, Li T, Du F, Ren J, Wu YT, Grishin NV, Chen ZJ (2015) Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science 347:aaa2630

    Article  PubMed  Google Scholar 

  49. Lee YJ, Park CK, Nam E, Kim SH, Lee OS, du Lee S, Lee C (2010) Generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus. J Virol Methods 163:410–415

    Article  CAS  PubMed  Google Scholar 

  50. Xu YL, Wu SP, Li YG, Sun FX, Wang QJ, Zhao Q, Yu J, Tian FL, Wu JQ, Zhu RL, Peng J (2020) A porcine alveolar macrophage cell line stably expressing CD163 demonstrates virus replication and cytokine secretion characteristics similar to primary alveolar macrophages following PRRSV infection. Vet Microbiol 244:108690

    Article  CAS  PubMed  Google Scholar 

  51. Chen XX, Qiao S, Li R, Wang J, Li X, Zhang G (2023) Evasion strategies of porcine reproductive and respiratory syndrome virus. Front Microbiol 14:1140449

    Article  PubMed  PubMed Central  Google Scholar 

  52. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock DT, Coyle AJ, Liao SM, Maniatis T (2003) IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 4:491–496

    Article  CAS  PubMed  Google Scholar 

  53. Al Hamrashdi M, Brady G (2022) Regulation of IRF3 activation in human antiviral signaling pathways. Biochem Pharmacol 200:115026

    Article  CAS  PubMed  Google Scholar 

  54. Deng M, Tam JW, Wang L, Liang K, Li S, Zhang L, Guo H, Luo X, Zhang Y, Petrucelli A, Davis BK, Conti BJ, June Brickey W, Ko CC, Lei YL, Sun S, Ting JP (2020) TRAF3IP3 negatively regulates cytosolic RNA induced anti-viral signaling by promoting TBK1 K48 ubiquitination. Nat Commun 11:2193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Cui J, Li Y, Zhu L, Liu D, Songyang Z, Wang HY, Wang RF (2012) NLRP4 negatively regulates type I interferon signaling by targeting the kinase TBK1 for degradation via the ubiquitin ligase DTX4. Nat Immunol 13:387–395

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Diao F, Bai J, Jiang C, Sun Y, Gao Y, Nauwynck H, Jiang P, Liu X (2023) The papain-like protease of porcine reproductive and respiratory syndrome virus impedes STING translocation from the endoplasmic reticulum to the Golgi Apparatus by deubiquitinating STIM1. J Virol 97:e0018823

    Article  PubMed  Google Scholar 

  57. Zhu Z, Zhang M, Yuan L, Xu Y, Zhou H, Lian Z, Liu P, Li X (2023) LGP2 promotes type I interferon production to inhibit PRRSV infection via enhancing MDA5-mediated signaling. J Virol 97:e0184322

    Article  PubMed  Google Scholar 

  58. Sun Z, Chen Z, Lawson SR, Fang Y (2010) The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions. J Virol 84:7832–7846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Professor Hanchun Yang from China Agricultural University for providing PRRSV strain BJ-4.

Funding

This study was supported by grants from the National Natural Science Foundation of China (32272993 and 32172875), the Program for Central Plains Youth Top Talent, and the Special Fund for Henan Agriculture Research System (HARS-22–12-S).

Author information

Authors and Affiliations

  1. Key Laboratory of Applied Technology On Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection & Internet Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, Hangzhou, 311300, Zhejiang, China

    Shuang-shuang Zhao

  2. Institute for Animal Health (Key Laboratory of Animal Immunology), Henan Academy of Agricultural Sciences, Zhengzhou, 450002, Henan, China

    Shuang-shuang Zhao, Qisheng Qian, Yao Wang, Songlin Qiao & Rui Li

Authors
  1. Shuang-shuang Zhao
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Qisheng Qian
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Yao Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Songlin Qiao
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Rui Li
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

RL and SQ jointly supervised the work. S-SZ and RL designed the experiments. S-SZ performed the studies. S-SZ and RL analysed the data. QQ and YW provided technical assistance. S-SZ wrote the manuscript. S-SZ, RL, and SQ revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Songlin Qiao or Rui Li.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

Additional information

Handling editor: Marie Galloux.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, Ss., Qian, Q., Wang, Y. et al. Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation. Vet Res 55, 151 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-024-01392-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-024-01392-w

Keywords

Veterinary Research

ISSN: 1297-9716