The A137R Protein of African Swine Fever Virus Inhibits Type I Interferon Production via the Autophagy-Mediated Lysosomal Degradation of TBK1

ABSTRACT African swine fever is a lethal hemorrhagic disease of pigs caused by African swine fever virus (ASFV), which greatly threatens the pig industry in many countries. Deletion of virulence-associated genes to develop live attenuated ASF vaccines is considered to be a promising strategy. A recent study has revealed that the A137R gene deletion results in ASFV attenuation, but the underlying mechanism remains unknown. To elucidate the mechanism of the A137R gene regulating ASFV virulence, an ASFV mutant with the A137R gene deleted (ASFV-ΔA137R) was generated based on the wild-type ASFV HLJ/2018 strain (ASFV-WT). Using transcriptome sequencing analysis, we found that ASFV-ΔA137R induced higher type I interferon (IFN) production in primary porcine alveolar macrophages (PAMs) than did ASFV-WT. Overexpression of the A137R protein (pA137R) inhibited the activation of IFN-β or IFN-stimulated response element. Mechanistically, pA137R interacts with TANK-binding kinase 1 (TBK1) and promotes the autophagy-mediated lysosomal degradation of TBK1, which blocks the nuclear translocation of interferon regulator factor 3, leading to decreased type I IFN production. Taken together, our findings clarify that pA137R negatively regulates the cGAS-STING-mediated IFN-β signaling pathway via the autophagy-mediated lysosomal degradation of TBK1, which highlights the involvement of pA137R regulating ASFV virulence. IMPORTANCE African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. Several virulence-associated genes of ASFV have been identified, but the underlying attenuation mechanisms are not clear. Compared with the virulent parental ASFV, the A137R gene-deleted ASFV mutant promoted the expression of type I interferon (IFN) in primary porcine alveolar macrophages. Further analysis indicated that the A137R protein negatively regulated the cGAS-STING-mediated IFN-β signaling pathway through targeting TANK-binding kinase 1 (TBK1) for autophagy-mediated lysosomal degradation. This study not only facilitates the understanding of ASFV immunoevasion strategies, but also provides new clues to the development of live attenuated ASF vaccines.

infected with either ASFV-DA137R or ASFV-WT. To determine the expected deletion of the A137R gene, the genome of ASFV-DA137R was analyzed by next-generation sequencing (NGS). Compared with the parental ASFV-WT, no undesirable variations were found in the genome of ASFV-DA137R, except a 391-bp deletion of the A137R gene replaced by the p72EGFP expression cassette. We then analyzed the effect of the A137R gene deletion on viral growth in vitro. PAMs were infected with ASFV-DA137R or ASFV-WT at a multiplicity of infection (MOI) of 0.01, and the viral titers were assayed by 50% hemadsorption doses (HAD 50 ) at the indicated time points. The growth profile of ASFV-DA137R was similar to that of ASFV-WT in PAMs (Fig. 1C).
The virulence of ASFV depends on the viral replication efficiency in target cells or the blockage of the immune responses by viral proteins. Since the A137R gene deletion does not influence the replication of ASFV-WT in vitro, we thus systemically analyzed the DEGs of the PAMs infected with ASFV-DA137R or ASFV-WT (MOI = 1) by RNA-seq analysis. Compared with the ASFV-WT-infected PAMs, the transcription levels of 2,458, 3,676, and 1,671 genes were altered upon ASFV-DA137R infection at 4, 12, and 20 hours post-infection (hpi), respectively (Fig. 1D). Notably, ASFV-DA137R infection induced higher type I IFN production in PAMs at 12 and 20 hpi, including IFN-A, IFN-b, and IFNv, than did ASFV-WT (Fig. 1E). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the upregulated genes upon ASFV-DA137R infection were involved in the IFN signaling pathway, such as cytosolic DNA-sensing, JAK-STAT, and Toll-like receptor (TLR) signaling pathways (Fig. 1F).
To verify the roles of pA137R regulation of the IFN signaling pathway, the transcription level of IFN-b was examined by reverse transcription-quantitative PCR (RT-qPCR) in the ASFV-DA137R-or ASFV-WT-infected PAMs. As shown in Fig. 2A, ASFV-DA137R, but not ASFV-WT, significantly enhanced IFN-b transcription in the PAMs at 12 to 20 hpi but not at 4 hpi, which was consistent with the RNA-seq analysis. Sendai virus (SeV), an IFN agonist, activates the retinoic acid inducible gene (RIG-I)-mediated IFN signaling pathway (24). ASFV-DA137R also induced higher transcription levels of IFN-A, IFN-b, and downstream IFNstimulated genes (ISGs), including ISG54 and ISG56, in the PAMs upon SeV stimulation than did ASFV-WT ( Fig. 2B to E). Taken together, these data indicate that pA137R inhibits the IFN signaling pathway and plays an important role in evading innate immune response.
pA137R inhibits the cGAS-STING-mediated IFN-b signaling pathway. The cGAS-STING-mediated IFN-b signaling pathway is significantly suppressed upon the virulent ASFV Armenia/07 infection, but the viral proteins involved remain elusive (5). The RNAseq analysis revealed that the cytosolic DNA-sensing signaling pathway was activated in the ASFV-DA137R-infected PAMs, and thus we further investigated the effects of pA137R on the cGAS-STING pathway. Human embryonic kidney (HEK293T) cells were cotransfected with p3ÂFlag-cGAS and -STING and reporter plasmids, along with pMyc-A137R or pCMV-Myc, for 24 h. The results indicated that overexpression of pA137R significantly inhibited the activation of the IFN-b or IFN-stimulated response element (ISRE) promoter in a dose-dependent manner ( Fig. 3A and B). Similarly, pA137R also markedly suppressed the transcription levels of IFN-b, ISG54, and ISG56 (Fig. 3C). These data suggest that pA137R negatively regulates the cGAS-STING-mediated IFN-b signaling pathway.
pA137R inhibits the IFN-b signaling pathway through targeting TBK1. To identify which adaptors in the cGAS-STING pathway were antagonized by pA137R, HEK293T cells were cotransfected with pMyc-A137R and reporter plasmids along with p3ÂFlag-cGAS, -STING, -TBK1, or -IRF3-5D (the active form of IRF3), respectively. We showed that pA137R markedly suppressed the activation of the IFN-b promoter induced by cGAS, STING, or TBK1, but not IRF3-5D (Fig. 4A to D). Therefore, we speculated that pA137R may inhibit the activation of the IFN-b promoter through targeting TBK1. Glutathione S-transferase (GST) pulldown assay indicated that pA137R specifically interacted with TBK1 but not with other adaptors, including cGAS, STING, or IRF3, of the cGAS-STING pathway (Fig. 5A). The colocalization of pA137R with TBK1 was observed in the cytoplasm, with a colocalization coefficient of 0.74 (Fig. 5B). PAMs were infected with ASFV-WT for 48 h, and the endogenous TBK1 was subjected to coimmunoprecipitation (co-IP) assay using anti-pA137R polyclonal antibodies (PAbs). As shown in Fig. 5C, TBK1 was precipitated by pA137R in the ASFV-infected PAMs. The colocalization of pA137R and TBK1 (a colocalization coefficient of 0.75) was further demonstrated by laser confocal microscopy (Fig. 5D). Collectively, these results indicate that pA137R blocks the IFN-b signaling pathway by interacting with TBK1.
pA137R promotes the autophagy-mediated lysosomal degradation of TBK1. Previous studies have revealed that the IFN signaling pathway is antagonized by several viruses, including human cytomegalovirus (HCMV) (25), Zika virus (ZIKV) (26), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (27), through targeting or degrading the host proteins. Since pA137R interacts with TBK1 and inhibits the activation of IFN-b ( Fig. 3 to 5), we investigated the effects of pA137R on the transcription and expression of TBK1. p3ÂFlag-TBK1 and pMyc-A137R were cotransfected into HEK293T cells. The results showed that TBK1 protein expression was inhibited by overexpression of pA137R in a dose-dependent manner, while the mRNA level of TBK1 remained unchanged ( Fig. 6A and B), indicating that pA137R may degrade the TBK1 protein. We further analyzed the TBK1 expression in the PAMs infected with ASFV-DA137R or ASFV-WT at 24 hpi. We found that ASFV-WT downregulated the expression  , and p3ÂFlag-cGAS (0.01 mg) and -STING (0.05 mg), along with pMyc-A137R (0.1, 0.25, or 0.5 mg) for 24 h followed by luciferase reporter assay. The expression of cGAS, STING, or pA137R was analyzed by Western blotting using mouse anti-Flag or -Myc monoclonal antibody. GAPDH was used as a loading control. (C) pA137R inhibits the mRNA levels of IFN-b and IFNstimulated genes (ISGs). HEK293T cells were cotransfected with p3ÂFlag-cGAS and -STING along with pMyc-A137R (0.5 mg) or pCMV-Myc (0.5 mg) for 24 h, and the mRNA levels of IFN-b, ISG54, and ISG56 were quantified by reverse transcription-quantitative PCR. Error bars denote standard errors of the means. All the data were analyzed using Student's t test: **, P , 0.01; ***, P , 0.001. of TBK1, while the TBK1 expression was significantly increased upon ASFV-DA137R infection in PAMs, although it was lower than that in the uninfected PAMs (Fig. 6C). Degradation of cellular proteins mainly relies on the ubiquitin-proteasome, autophagosome, or lysosome pathway. Therefore, HEK293T cells were cotransfected with p3ÂFlag-TBK1 and pMyc-A137R for 24 h and then treated with proteasome inhibitor MG132, autophagosome inhibitor 3-methyladenine (3-MA), or lysosomal inhibitor bafilomycin A1 (BafA1), respectively, for 8 h. None of the inhibitors affected the viability of HEK293T cells (Fig. 6D), and the pA137Rmediated TBK1 degradation was inhibited by BafA1 or 3-MA, but not MG132 (Fig. 6E). Since autophagy-related protein 5 (ATG5) is essential for autophagosome formation (28), we therefore examined the involvement of autophagosome in the degradation of TBK1 by , along with pMyc-A137R or pCMV-Myc, and the luciferase activities were measured at 24 hours post-infection. The expression of cGAS, STING, TBK1, IRF3-5D, or pA137R was analyzed by Western blotting using mouse anti-Flag or -Myc monoclonal antibody. GAPDH was used as a loading control. Error bars denote standard errors of the means. All the data were analyzed using Student's t test: ***, P , 0.001; ns, not significant. pA137R using the ATG5-knockout HeLa cells. Consistent with the inhibitor treatment assay, the degradation of TBK1 by pA137R was counteracted in the ATG5-knockout HeLa cells compared with the parental wild-type (WT) cells (Fig. 6F). Altogether, these results indicate that the autophagy-mediated lysosomal pathway is responsible for the degradation of TBK1 by pA137R.
pA137R blocks the nuclear translocation of IRF3. With the activation of the cGAS-STING pathway, the activated IRF3 is translocated into the nucleus and triggers IFN-b transcription. Since IRF3 is activated by TBK1, we speculated that TBK1 degradation may influence IRF3 translocation from the cytoplasm into the nucleus. HEK293T cells were transfected with pMyc-A137R or pCMV-Myc for 24 h and then stimulated FIG 5 The A137R protein (pA137R) interacts with TBK1. (A) HEK293T cells were transfected with p3ÂFlag-cGAS, -STING, -TBK1, or -IRF3 for 48 h and lysed with NP-40 buffer. The purified GST or GST-pA137R protein was used to pull down the key adaptors of the cGAS-STING pathway in the lysates and analyzed by Western blotting using mouse anti-GST or -Flag monoclonal antibody (MAb). (B) HEK293T cells were cotransfected with p3ÂFlag-TBK1 and pMyc-A137R for 24 h and then incubated with rabbit anti-Flag or mouse anti-Myc MAb and Alexa Fluor 488 (green)-or 633 (red)-conjugated secondary antibody, respectively. Cell nuclei (blue) were stained with 49,6-diamidino-2-phenylindole (DAPI). Bars, 10 mm. (C) Primary porcine alveolar macrophages (PAMs) were infected with the wild-type ASFV HLJ/2018 strain (ASFV-WT) at a multiplicity of infection (MOI) of 1 for a coimmunoprecipitation assay. The lysates were collected at 48 hours post-infection and incubated with protein G agarose, along with rabbit anti-pA137R polyclonal antibodies (PAbs) or irrelevant rabbit immunoglobulin G (IgG), and then the bound proteins were analyzed by Western blotting using in-house mouse anti-pA137R or rabbit anti-TBK1 PAbs. (D) PAMs were uninfected or infected with ASFV-WT for 24 h at an MOI of 1, and the localization of pA137R and TBK1 was visualized by laser confocal microscopy using in-house mouse anti-pA137R or rabbit anti-TBK1 PAbs and the indicated secondary antibody, respectively. Cell nuclei (blue) were stained with DAPI as described above. Bars, 10 mm. , respectively. The expression of TBK1 or pA137R was analyzed as described above. GAPDH was used as a loading control. (F) pMyc-A137R and p3ÂFlag-TBK1 were cotransfected into autophagy-related protein 5 (ATG5)-knockout or wild-type (WT) HeLa cells for 36 h, and the expression of TBK1 or pA137R was examined with the indicated antibodies as described above. GAPDH was used as a loading control. Error bars denote standard errors of the means. All the data were analyzed using Student's t test. ns, not significant.
with SeV or poly(dA-dT). The subcellular localization of IRF3 in the absence or presence of pA137R was determined by laser confocal microscopy ( Fig. 7A and B). In the unstimulated group, the IRF3 was mainly located in the cytoplasm. Upon SeV or poly(dA-dT) stimulation, approximately 30 to 40% of IRF3 translocated into the nucleus in the pCMV-Myc-transfected cells. However, only 10% of IRF3 was detected in the nucleus when there was ectopic expression of pA137R. Thus, pA137R degrades TBK1, leading to blockage of the nuclear translocation of IRF3.

DISCUSSION
Previous studies have shown that knocking out the immunoregulatory genes usually alters ASFV virulence (8-11, 17, 29-31). pA137R is a late-expressed viral structural protein and associated with ASFV virulence (20,32), but the underlying attenuation mechanism is not clear. In this study, we found that ASFV-DA137R significantly increased type I IFN production in PAMs compared with ASFV-WT ( Fig. 1 and 2). pA137R suppressed the cGAS-STING-mediated IFN-b signaling pathway through targeting TBK1 (Fig. 3 and 4). Furthermore, we demonstrated that pA137R interacts with TBK1, promotes its degradation via the autophagy-mediated lysosomal pathway, and affects IRF3 nuclear translocation to block type I IFN production (Fig. 8). To our knowledge, this is the first study to elaborate the mechanism of pA137R regulation of ASFV virulence.
TBK1 undergoes autophosphorylation and ubiquitination to facilitate the activation of downstream adaptors (e.g., IRF3 or NF-k B) during viral infections. However, viral proteins target TBK1 to downregulate IFN production by disrupting TBK1-associated complexes or degrading TBK1: e.g., the HCMV UL94 protein (25), the herpes simplex virus 1 (HSV-1) US11 (33) and g34.5 (34) proteins, the foot-and-mouth disease virus L pro protein (35), the swine acute diarrhea syndrome coronavirus nucleocapsid protein (36), the heartland virus nonstructural proteins (37), and the SARS-CoV-2 M protein (38). In this study, we found that type I IFN production was inhibited by pA137R in PAMs upon ASFV infection ( Fig. 1 and 2), and the TBK1 expression was also downregulated in the HEK293T cells with pA137R-overexpressed or ASFV-WT-infected PAMs (Fig. 6). TBK1, a critical adaptor of the cGAS-, RIG-I-, and melanoma differentiation-associated antigen 5 (MDA-5)-mediated signaling pathways (39), is degraded by ubiquitin-specific protease 38 (USP38) (40), major vault protein (MVP) (41), and dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) (42) via the proteasomal or autophagy-mediated lysosomal pathway. It has been revealed that the ASFV DP96R protein degrades TBK1 (14), but the underlying mechanism remains unknown. We have demonstrated that pA137R interacts with and degrades TBK1 via the autophagy-mediated lysosomal pathway (Fig. 5 and 6). The expression of TBK1 in the ASFV-DA137R-infected PAMs was higher than that in the ASFV-WT-infected PAMs but lower than that in the uninfected PAMs, implying that other ASFV proteins besides pA137R might also degrade TBK1.  (47) to trigger the activation of the IFN signaling pathway. To evade innate immune responses, pA137R interacts with and degrades TBK1 via the autophagy-mediated lysosomal pathway, followed by the inhibition of IRF3 nuclear translocation to suppress the IFN-b production upon ASFV infection.
It is generally accepted that the early-expressed viral proteins play a key role in regulating IFN production, but several studies have shown that the late-expressed viral proteins are also involved in this process and associated with viral virulence. UL94, a late-expressed protein and structural component of HCMV, inhibits IFN-b production by disrupting the dimerization of MITA and recruitment of TBK1 to MITA (25,51,52). The HSV-1 US11 protein interacts with RIG-I and MDA-5 and antagonizes IFN-b production (50); the herpesviral g34.5 protein prevents protein synthesis via the protein kinase R (PKR) signaling pathway or inhibits the production of IFN-b by promoting TBK1 degradation and disrupting the interaction between TBK1 and IRF3 (33,53). IFN-b inhibits viral infections, and low-virulence viruses in turn increase IFN-b production, such as West Nile virus (54), Japanese encephalitis virus (55), bluetongue virus (56), and ASFV (6)(7)(8)(9)(10)(11). Deletion of the IFN antagonist from the virulent ASFV results in attenuation (7)(8)(9)(10)(11). The A137R gene is associated with the virulence of ASFV (20), its deletion resulted in more production of IFN-a, IFN-b, ISG54, and ISG56 in PAMs ( Fig. 1 and 2).
There is an urgent need to define the determinants of ASFV virulence, which contributes to the development of treatments and vaccines. A low dose (10 2 HAD 50 ) of the A137R gene-deleted ASFV mutant (ASFV-G-DA137R) results in ASFV attenuation and offers protection against challenge with the virulent parental ASFV-G, but the ASFV-G-DA137R-inoculated pigs present high viremia titers (10 5 to 10 6 ) and shed the virus (20). It has been reported that pA137R is highly conserved among the genotype I and II strains (20). Because the attenuation of ASFV is relevant to the increased production of IFN (7)(8)(9)(10)(11) and ASFV-DA137R promotes IFN-b production in PAMs, we speculate that pA137R promotes the autophagy-mediated lysosomal degradation of TBK1 to negatively regulate IFN-b production and attenuate ASFV virulence. However, we cannot rule out the differences in the genetic backgrounds of virus strains, which lead to changes in phenotypes. Deletion of EP402R from the virulent BA71 strain results in attenuation, whereas the phenotype is not observed in the ASFV Georgia 2007 and HLJ/2018 strains (22,(57)(58)(59). Deletion of DP148R from the Benin 97/1 strain leads to ASFV attenuation (60), but not the ASFV HLJ/2018 strain (59), which was isolated in China (58). However, the UK/CD2v-deleted ASFV strain also presents different attenuation in these strains (59,61).
In conclusion, our research is the first to reveal that pA137R interacts with and degrades TBK1 via the autophagy-mediated lysosomal pathway to inhibit IFN-b production. These findings enrich the understanding of immunoevasion by ASFV and the mechanism of pA137R regulation of ASFV virulence.
Dual-luciferase reporter (DLR) assays. HEK293T cells were cotransfected with reporter plasmids (pIFN-b-Fluc or pISRE-Fluc [0.05 mg]) and pTK-Rluc (0.01 mg), along with or without the indicated expression plasmids, using X-tremeGENE HP (catalog no. 6366546001; Roche). After 24 h of transfection, the cells were washed with phosphate-buffered saline (PBS) three times. The passive lysis buffer was added for 20 min at 4°C with gentle shaking, and then the luciferase activities were determined using the DLR assay system (catalog no. E1910; Promega) according to the manufacturer's instructions. The data are represented as the ratio of Fluc to Rluc.
Generation of the ASFV-DA137R mutant. To generate the ASFV-DA137R mutant by homologous recombination, the transfer vector was constructed, which harbors the p72p72 promoter-controlled EGFP gene and the left and right homologous arms of the A137R gene that are located in nucleotides (nt) 52692 to 54570 and 54962 to 55960, respectively, of the ASFV-WT genome. The nucleotides in the genome from positions 54571 to 54961 were replaced by the p72EGFP expression cassette. Briefly, the left and right homologous arms of the A137R gene with restriction enzyme sites were amplified using  the gDNA of ASFV-WT as the template, and then fused to the p72EGFP expression cassette by overlapping PCR using the primers listed in Table 1, and the resulting fusion fragment was cloned into the pOK-12 vector. The transfer vector was sequenced and named pOK-p72-DA137R-EGFP. The ASFV-DA137R mutant was generated by homologous recombination according to the previously described methods (61). Briefly, PAMs were seeded in 6-well plates coated with poly-L-lysine and incubated for 24 h at 37°C. The PAMs were transfected with 2 mg of transfer vector pOK-p72-DA137R-EGFP using X-tremeGENE HP for 16 h and then infected with ASFV-WT (MOI = 3). The recombinant viruses in PAMs were harvested until the EGFP expression, followed by purification using limiting dilution.
NGS. To confirm the accurate deletion of the A137R gene in the ASFV genome, the gDNA of ASFV-DA137R was extracted from the infected PAMs using the QIAamp blood mini kit (catalog no. 51104; Qiagen), and the full-length sequence of the ASFV-DA137R genome was determined by NGS as described previously (66).
RNA-seq analysis. PAMs were infected with ASFV-DA137R or ASFV-WT at an MOI of 1. At 4, 12, and 20 hpi, the cells were used to extract total RNAs by using TRIzol reagent (catalog no. 15596026; Invitrogen) according to the manufacturer's instructions. RNA quantification and qualification were assessed using the RNA Nano 6000 assay kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The mRNAs with poly(A) tails were enriched and purified from the total mRNAs by poly(T) oligonucleotide-coupled magnetic beads. RNA-seq libraries were prepared and assessed by AMPure XP system kit (Beckman Coulter, Beverly, USA) in the Agilent Bioanalyzer 2100 system. Subsequently, the RNA-seq libraries were sequenced on an Illumina Novaseq platform (Nova gene, China). KEGG enrichment analysis of DEGs was performed using Cluster Profiler (3.4.4).
RNA extraction and RT-qPCR. The total RNAs of the cells with indicated treatment or virus infection were extracted with the Simply P total RNA extraction kit (catalog no. BSC52M1; BioFlux) and transcribed into cDNA by FastKing gDNA Dispelling RT SuperMix (catalog no. KR118-02; Tiangen). The cDNAs from HEK293T cells or PAMs were used as templates for RT-qPCR using SYBR Premix Ex Taq (catalog no. RR390B; TaKaRa) to examine the mRNA level of human IFN-b, ISG54, ISG56, or TBK1, and porcine IFN-a, IFN-b, ISG54, or ISG56, respectively. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal reference, and all the primers are listed in Table 1.
Co-IP assay. PAMs were infected with ASFV-WT at an MOI of 1, and the lysates were collected at 48 hpi for the co-IP assay. Protein G agarose (catalog no. 11243233001; Roche) was incubated with the lysates and rabbit anti-pA137R PAbs or a rabbit immunoglobulin G (catalog no. ab190492; Abcam) (isotype control antibody). The bound proteins were subjected to Western blotting using rabbit anti-TBK1 (catalog no. 3504S; Cell Signaling Technology) and in-house mouse anti-pA137R PAbs.
Cell viability assay. Cell viability was determined by Cell Counting Kit-8 (catalog no. K1018; APExBIO) according to the manufacturer's instructions.
Statistical analysis. All data were determined in triplicates and analyzed with SPSS 22.0 software (SPSS Software, Inc.). Student's t test was used to assess statistical significance. A P value of ,0.05 was considered as significant.