African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D
2021; Elsevier BV; Volume: 298; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2021.101480
ISSN1083-351X
AutoresGaihong Zhao, Tingting Li, Xuemin Liu, Taoqing Zhang, Zhaoxia Zhang, Kang Li, Jie Song, Shijun Zhou, Xin Chen, Xiao Wang, Jiangnan Li, Li Huang, Changyao Li, Zhigao Bu, Jun Zheng, Changjiang Weng,
Tópico(s)Vector-Borne Animal Diseases
ResumoAfrican swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N1–107). Interestingly, unlike the effect of GSDMD-N1–279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N1–107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication. African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N1–107). Interestingly, unlike the effect of GSDMD-N1–279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N1–107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication. African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is characterized by a series of different clinical symptoms, including anorexia, moribundity, bloody diarrhea, vomiting, high fever, and hemorrhagic symptoms (1Dixon L.K. Sun H. Roberts H. African swine fever.Antiviral Res. 2019; 165: 34-41Crossref PubMed Scopus (132) Google Scholar, 2Sánchez-Cordón P. Montoya M. Reis A. Dixon L. African swine fever: A re-emerging viral disease threatening the global pig industry.Vet. J. 2018; 233: 41-48Crossref PubMed Scopus (180) Google Scholar). Because ASF was first discovered in Kenya in the 1920s, it has rapidly spread to many areas in sub-Saharan Africa, the Caucasus, and Eastern Europe (2Sánchez-Cordón P. Montoya M. Reis A. Dixon L. African swine fever: A re-emerging viral disease threatening the global pig industry.Vet. J. 2018; 233: 41-48Crossref PubMed Scopus (180) Google Scholar). The first ASF case was reported in August of 2018 in China and caused serious economic losses (3Wang T. Sun Y. Huang S. Qiu H.J. Multifaceted immune responses to African swine fever virus: Implications for vaccine development.Vet. Microbiol. 2020; 249: 108832Crossref PubMed Scopus (10) Google Scholar). To date, there are no effective drugs or commercial vaccines against American swine fever virus (ASFV) (4Teklue T. Sun Y. Abid M. Luo Y. Qiu H.J. Current status and evolving approaches to African swine fever vaccine development.Transbound. Emerg. Dis. 2020; 67: 529-542Crossref PubMed Scopus (33) Google Scholar). ASFV is the only member of the family Asfarviridae, genus Asfivirus. The ASFV virion is a large, icosahedral structure of approximately 200 nm. The ASFV genome contains a linear double-stranded DNA (170–190 kbp) that encodes approximately 150 proteins (5Alejo A. Matamoros T. Guerra M. Andrés G. A proteomic atlas of the African swine fever virus particle.J. Virol. 2018; 92e01293-18Crossref PubMed Scopus (101) Google Scholar). Similar to other nucleocytoplasmic large DNA viruses (NCLDVs), ASFV encodes many proteins involved in not only the viral life cycle, including viral entry, DNA replication and repair, viral assembly, and egress (6Alonso C. Galindo I. Cuesta-Geijo M.A. Cabezas M. Hernaez B. Muñoz-Moreno R. African swine fever virus-cell interactions: From virus entry to cell survival.Virus Res. 2013; 173: 42-57Crossref PubMed Scopus (37) Google Scholar) but also the evasion of host defenses, including the inhibition of host innate immune responses (such as type I-interferon production and inflammatory responses) and cell death pathways (7Reis A.L. Netherton C. Dixon L.K. Unraveling the armor of a killer: Evasion of host defenses by African swine fever virus.J. Virol. 2017; 91e02338-16Crossref PubMed Scopus (43) Google Scholar). ASFV encodes two large polyprotein precursors, pp220 and pp62, which are proteolytically cleaved by ASFV pS273R to yield the structural proteins required for virus assembly (8Andrés G. Alejo A. Salas J. Salas M.L. African swine fever virus polyproteins pp220 and pp62 assemble into the core shell.J. Virol. 2002; 76: 12473-12482Crossref PubMed Scopus (50) Google Scholar, 9Andrés G. Alejo A. Simón-Mateo C. Salas M.L. African swine fever virus protease, a new viral member of the SUMO-1-specific protease family.J. Biol. Chem. 2001; 276: 780-787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The cysteine protease pS273R encoded by ASFV is synthesized at the late stages of viral infection and is localized within cytoplasmic viral factories (9Andrés G. Alejo A. Simón-Mateo C. Salas M.L. African swine fever virus protease, a new viral member of the SUMO-1-specific protease family.J. Biol. Chem. 2001; 276: 780-787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The overall structure of the ASFV pS273R is represented by two domains named the N-terminal "arm domain" and the "core domain" (10Li G. Liu X. Yang M. Zhang G. Wang Z. Guo K. Gao Y. Jiao P. Sun J. Chen C. Wang H. Deng W. Xiao H. Li S. Wu H. et al.Crystal structure of African swine fever virus pS273R protease and implications for inhibitor design.J. Virol. 2020; 94e02125-19Crossref PubMed Scopus (9) Google Scholar). The "arm domain" contains the residues from M1 to N83, which is unique to ASFV and plays an important role in maintaining the enzyme activity of ASFV pS273R (10Li G. Liu X. Yang M. Zhang G. Wang Z. Guo K. Gao Y. Jiao P. Sun J. Chen C. Wang H. Deng W. Xiao H. Li S. Wu H. et al.Crystal structure of African swine fever virus pS273R protease and implications for inhibitor design.J. Virol. 2020; 94e02125-19Crossref PubMed Scopus (9) Google Scholar). The "core domain" contains the residues from N84 to A273, which shares a high degree of structural similarity with chlamydial-deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease. Pyroptosis is a recently discovered form of programmed cell death that is activated in response to diverse microbial ligands, including bacterial flagellin, toxins, lipopolysaccharide, and DNA that gains access to the cell cytosol (11Broz P. Dixit V.M. Inflammasomes: Mechanism of assembly, regulation and signalling.Nat. Rev. Immunol. 2016; 16: 407-420Crossref PubMed Scopus (1241) Google Scholar). Pyroptosis is characterized by the activation of inflammatory caspases (such as caspase-1 and caspase-4/5/11) and pore formation in the cellular plasma membrane, resulting in the release of a large number of proinflammatory cytokines (12Jorgensen I. Miao E. Pyroptotic cell death defends against intracellular pathogens.Immunol. Rev. 2015; 265: 130-142Crossref PubMed Scopus (466) Google Scholar). Recently, gasdermin family members, such as gasdermin D (GSDMD), were found to be cleaved by inflammatory-related caspases to execute pyroptosis. For example, GSDMD, a critical mediator of pyroptosis, can be cleaved within a linker between the N-terminal domain and the C-terminal domain by activated caspase-1 (13He W.T. Wan H. Hu L. Chen P. Wang X. Huang Z. Yang Z.H. Zhong C.Q. Han J. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.Cell Res. 2015; 25: 1285-1298Crossref PubMed Scopus (859) Google Scholar, 14Shi J. Zhao Y. Wang K. Shi X. Wang Y. Huang H. Zhuang Y. Cai T. Wang F. Shao F. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.Nature. 2015; 526: 660-665Crossref PubMed Scopus (2095) Google Scholar), caspase-4/5/11 (15Kayagaki N. Warming S. Lamkanfi M. Vande Walle L. Louie S. Dong J. Newton K. Qu Y. Liu J. Heldens S. Zhang J. Lee W. Roose-Girma M. 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Subsequently, the N-terminal domain of human GSDMD (GSDMD-N1–275) oligomerizes to be inserted into the plasma membrane, resulting in pore formation (19Liu X. Zhang Z. Ruan J. Pan Y. Magupalli V.G. Wu H. Lieberman J. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.Nature. 2016; 535: 153-158Crossref PubMed Scopus (1121) Google Scholar). It is widely accepted that the pore formation by human GSDMD-N1–275 results in the loss of osmotic homeostasis, leading to cell swelling and death, which releases inflammatory factors to inhibit and clear intracellular pathogens (20Xia S. Hollingsworth L.R.T. Wu H. Mechanism and regulation of gasdermin-mediated cell death.Cold Spring Harb. Perspect. Biol. 2020; 12a036400Crossref PubMed Scopus (39) Google Scholar, 21Xia S. Ruan J. Wu H. Monitoring gasdermin pore formation in vitro.Methods Enzymol. 2019; 625: 95-107Crossref PubMed Scopus (9) Google Scholar). Recently, the caspase-3-mediated cleavage of GSDME (22Wang Y. Gao W. Shi X. Ding J. Liu W. He H. Wang K. Shao F. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.Nature. 2017; 547: 99-103Crossref PubMed Scopus (768) Google Scholar) and the granzyme A-mediated cleavage of GSDMB were also reported (23Zhou Z. He H. Wang K. Shi X. Wang Y. Su Y. Wang Y. Li D. Liu W. Zhang Y. Shen L. Han W. Shen L. Ding J. Shao F. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells.Science. 2020; 368eaaz7548Crossref PubMed Scopus (202) Google Scholar). Here, we report that swine GSDMD is a novel binding partner of the ASFV-encoded pS273R. ASFV infection decreased the expression of GSDMD by cleavage of GSDMD at the G107-A108 pair to yield a nonfunctional GSDMD-N fragment consisting of amino acids (aa) 1 to 107 (GSDMD-N1–107). Mechanistically, unlike the GSDMD-N1–279 produced by caspase-1, the GSDMD-N1–107 produced by pS273R did not induce pyroptosis and inhibit ASFV replication. Our findings reveal a previously unrecognized novel mechanism by which ASFV evades the host antiviral innate immune responses. To investigate the novel function of ASFV-encoded pS273R (ASFV pS273R) on the host cellular immune responses, we first screened and identified the host proteins that interact with ASFV pS273R by pull down-mass spectrometry. The cell lysates of porcine alveolar macrophages (PAMs) were incubated with His-pS273R protein bound to Ni Sepharose. ASFV pS273R-binding proteins were eluted and stained with Coomassie brilliant blue. We found that several specific bands were observed in the eluted proteins in the His-pS273R group (lane 3) compared with the control group (lane 1 and lane 2) (Fig. 1A). Then, the gels containing these specific bands (lane 3) were cut into three pieces as indicated and analyzed by mass spectrometry. As shown in Table 1, 12 pS273R-interacting proteins were identified in the sample 1# (sample 2# and 3# data not shown). Among them, GSDMD, a pyroptosis executor, was selected for subsequent studies due to its score and role in pyroptosis.Table 1Results of mass spectrometry analysis of the host proteins that interact with pS273RSample nameAccessionMass (Da)ScoreMatchesSequencesemPAIProtein description1GSDMD51,94161330 (21)12 (10)2.22Gasdermin D2ARHGAP45126,23527724 (11)20 (9)0.36Minor histocompatibility protein HA-1 isoform X33NOP970,3891608 (3)7 (2)0.15Uncharacterized protein4WDR7165,83711613 (4)9 (4)0.08WD repeat domain 75RUFY368,422604 (2)3 (1)0.1Protein RUFY3 isoform X46CHERP104,279535 (1)5 (1)0.03Calcium homeostasis endoplasmic reticulum protein7ABCE168,254397 (1)6 (1)0.05Uncharacterized protein8RPL448,287393 (2)2 (2)0.14Ribosomal protein L49ECE2172,699353 (1)3 (1)0.04Uncharacterized protein10FLII150,898312 (0)2 (0)0.02FLII actin remodeling protein11CRIP18926211 (0)1 (0)0.39Cysteine-rich intestinal protein 112STAT381,896207 (0)5 (0)0.04Signal transducer and activator of transcription Open table in a new tab To further characterize the interaction between ASFV pS273R and swine GSDMD, Flag-pS273R or HA-GSDMD alone or both were coexpressed in HEK293T cells, and the interaction and the subcellular colocalization of the two proteins were examined. As shown in Figure 1, B and C, ASFV pS273R interacted with and colocalized with GSDMD in the cytoplasm. In addition, we noticed that ASFV S273R interacted with the C-terminal of swine GSDMD and GSDMD in a low activity proteolysis. We also observed that the reduced abundance of full-length GSDMD (55 kDa) was accompanied by the appearance of an N-terminal fragment of GSDMD with apparent molecular masses of approximately 10 and 15 kDa (designated GSDMD-N), suggesting that ASFV pS273R may cleave swine GSDMD in vitro. To further confirm whether GSDMD is cleaved by ASFV pS273R, HEK293T cells were cotransfected with a plasmid encoding Flag-GSDMD and increasing amounts of a plasmid expressing ASFV pS273R. As shown in Figure 1D, the intensity of Flag-GSDMD diminished as the protein levels of ASFV pS273R were increased, and an N-terminal fragment of GSDMD began to appear, suggesting that GSDMD is cleaved by ASFV pS273R in a dose-dependent manner. To further confirm that ASFV pS273R directly cleaves GSDMD in vitro, purified ASFV pS273R (6 × His-pS273R) and GST-GSDMD were incubated, and the reaction products were detected with anti-His and anti-GSDMD antibodies. As shown in Figure 1E, we found that the purified ASFV pS273R cleaved GST-GSDMD and produced a GST-GSDMD-N fragment with a molecular mass of approximately 39 kDa. To further investigate whether GSDMD is specifically cleaved by ASFV pS273R, HEK293T cells were transfected with a plasmid expressing Flag-tagged GSDMA, GSDMB, GSDMC, GSDMD, or GSDME in combination with an empty vector or a plasmid expressing HA-pS273R. As shown in Figure 1F, GSDMD, but none of the other GSDMDs chosen in this study, was cleaved by ASFV pS273R. To test whether ASFV pS273R interacts with endogenous GSDMD in PAMs upon ASFV infection. PAMs were infected with ASFV HLJ/18 and a Co-IP assay was performed. We found that ASFV pS273R interacted with endogenous GSDMD in PAMs infected with ASFV (Fig. 2A). Meanwhile, we also noticed that the ASFV pS273R colocalized with endogenous GSDMD in the cytoplasm in PAMs following ASFV infection (Fig. 2B). These data suggested that ASFV pS273R specifically interacts with GSDMD. To investigate the impact of ASFV infection on the integrity of GSDMD, we analyzed the expression of GSDMD in PAMs either mock-infected or infected with ASFV at different multiplicities of infection (MOIs). As shown in Figure 2C, upon viral infection with increasing doses of ASFV, the levels of endogenous GSDMD decreased in a dose-dependent manner, and only an approximately 40 kDa cleavage product (designated GSDMD-C) was recognized by the anti-GSDMD antibody against the C-terminus of GSDMD. To explore whether the protease activity of ASFV pS273R is required for the cleavage of GSDMD, we evaluated the impact of Z-VAD-FMK, a pan-caspase inhibitor, on GSDMD cleavage by pS273R. As shown in Figure 3, A and B, Z-VAD-FMK did not affect ASFV pS273R-mediated GSDMD cleavage although it inhibited caspase-1-mediated GSDMD cleavage, suggesting that ASFV pS273R-mediated GSDMD cleavage was not dependent on the activities of host caspases. His168 and Cys232 are key amino acids that form the two catalytic sites of ASFV pS273R (9Andrés G. Alejo A. Simón-Mateo C. Salas M.L. African swine fever virus protease, a new viral member of the SUMO-1-specific protease family.J. Biol. Chem. 2001; 276: 780-787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). To further verify this issue, we constructed three plasmids expressing different mutant forms of ASFV pS273R, such as pS273R-H168R, C232S, and H168R/C232S (Fig. 3C), and used them to analyze GSDMD cleavage. As shown in Figure 3D, ASFV pS273R with H168R, C232S, or H168R/C232S (DM) substitutions within the active site disrupted its protease activity, resulting in loss of the cleavage of GSDMD. Hence, these results demonstrate that the protease activity of ASFV pS273R is essential for GSDMD cleavage. Previous reports demonstrated that ASFV pS273R preferentially cleaves Gly-Gly (G-G) amino acid pairs within pp62 and pp220 (9Andrés G. Alejo A. Simón-Mateo C. Salas M.L. African swine fever virus protease, a new viral member of the SUMO-1-specific protease family.J. Biol. Chem. 2001; 276: 780-787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). According to the features of the pS273R cleavage sites with pp62 and pp220 (Fig. 4A, above), we examined the amino acid sequence of the swine GSDMD for potential ASFV pS273R cleavage sites and found that four regions bearing several glycines (G) resemble the signature G-G sequences of the proteolytic sites of ASFV pS273R (Fig. 4A, bottom). Therefore, we inferred that at least four potential ASFV pS273R cleavage sites may exist in swine GSDMD. To define the putative cleavage site, we constructed a series of GSDMD mutants, in which the glycine was replaced with alanine (such as G78A, G107A, G320A, and G345A). GSDMD or its mutants were coexpressed with ASFV pS273R in HEK293T cells to identify the actual cleavage sites. As shown in Figure 4B, GSDMD-G107A was absolutely resistant to ASFV pS273R. However, other GSDMD mutants did not prevent ASFV pS273R cleavage. Compared with the human GSDMD sequence, we predicted that Asp279 (D279) is a site at which swine GSDMD is cleaved by caspase-1 (Fig. 4C). To further test whether G107 is indeed the cleavage site, we generated a GSDMD-G107A/D279A double mutant (GSDMD-DM). ASFV pS273R was coexpressed with GSDMD, GSDMD-G107A, GSDMD-D279A, and GSDMD-DM. As shown in Figure 4D, we found that GSDMD and GSDMD-D279A were still cleaved by ASFV pS273R. However, GSDMD-G107A and GSDMD-DM were completely resistant to ASFV pS273R cleavage. In contrast, GSDMD-D279A and GSDMD-DM were resistant to caspase-1 (Fig. 4E). Consistent with these results, we also noticed that GSDMD-C108–488 could be cleaved by caspase-1 (Fig. 4F). Taken together, these findings suggest that swine GSDMD is cleaved at G107 by ASFV pS273R and at D279 by caspase-1. Previous reports showed that the cleavage product (GSDMD-N1–275) of human GSDMD by active caspase-1 induces pyroptosis by forming a large pore in the cell plasma membrane (9Andrés G. Alejo A. Simón-Mateo C. Salas M.L. African swine fever virus protease, a new viral member of the SUMO-1-specific protease family.J. Biol. Chem. 2001; 276: 780-787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 19Liu X. Zhang Z. Ruan J. Pan Y. Magupalli V.G. Wu H. Lieberman J. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.Nature. 2016; 535: 153-158Crossref PubMed Scopus (1121) Google Scholar). We constructed two plasmids expressing swine GSDMD-N1–107 and GSDMD-C108–488 that mimic cleavage products generated by ASFV pS273R (Fig. 5A). We also constructed two other plasmids expressing swine GSDMD-N1–279 and GSDMD-C280–488 that mimic the cleavage products generated by active caspase-1. In addition, we constructed a plasmid expressing GSDMD-C108–279, a cleavage product of GSDMD by ASFV S273R and caspase-1. The expression levels of these proteins were verified (Fig. 5B). To visualize the cellular distribution of transiently expressed WT GSDMD and the cleavage products of GSDMD, HEK293T cells were transfected with a plasmid expressing GSDMD-WT and its cleaved fragments, including GSDMD-N1–107, GSDMD-C108–488, GSDMD-C280–488, GSDMD-C108–279, and GSDMD-N1–279, respectively. Alexa Fluor 594-conjugated wheat germ agglutinin was used to stain the plasma membrane. We found that GSDMD-WT and GSDMD-N1–107 diffusely localized in the cytosol and nuclear, GSDMD-C108–488, GSDMD-C280–488, and GSDMD-C108–279 localized in the cytosol whereas only GSDMD-N1–279 localized in the cell membrane (Fig. 5C). Consistent with these results, GSDMD-N1–279, but not GSDMD and other cleavaged fragments, induces cell death with typical pyroptosis morphological features (Fig. 5D). Furthermore, we found that only GSDMD-N1–279 induced LDH release (Fig. 5E) and decreased cell viability (Fig. 5F). Taken together, these results indicate that pS273R may cleave GSDMD to produce GSDMD-N1–107 to lose its function. Previous studies reported that several viral proteases can cleave GSDMD to affect pyroptosis, resulting in the regulation of viral replication (24Yamaoka Y. Matsunaga S. Jeremiah S.S. Nishi M. Miyakawa K. Morita T. Khatun H. Shimizu H. Okabe N. Kimura H. Hasegawa H. Ryo A. Zika virus protease induces caspase-independent pyroptotic cell death by directly cleaving gasdermin D.Biochem. Biophys. Res. Commun. 2021; 534: 666-671Crossref PubMed Scopus (6) Google Scholar, 25Lei X. Zhang Z. Xiao X. Qi J. He B. Wang J. Enterovirus 71 inhibits pyroptosis through cleavage of gasdermin D.J. Virol. 2017; 91e01069-17Crossref PubMed Scopus (52) Google Scholar). Therefore, we first tested whether the swine GSDMD cleavage products generated by ASFV pS273R and caspase-1 are involved in ASFV replication. The plasmids expressing GSDMD, GSDMD-N1–107, GSDMD-C108–488, GSDMD-N1–279, and GSDMD-C280–488, GSDMD-C108–279 were ectopically transfected into MA-104 cells (Fig. 6A), a suitable cell line for ASFV growth (26Rai A. Pruitt S. Ramirez-Medina E. Vuono E. Silva E. Velazquez-Salinas L. Carrillo C. Borca M. Gladue D. Identification of a continuously stable and commercially available cell line for the identification of infectious African swine fever virus in clinical samples.Viruses. 2020; 12: 820Crossref Scopus (12) Google Scholar). Meanwhile, the cells were infected with ASFV for 24 h, and ASFV genomic DNA was extracted for quantitative PCR analysis. As shown in Figure 6C, ASFV replication significantly decreased in the cells expressing GSDMD-N1–279. However, other cleavage products of GSDMD had no inhibitory effect on ASFV replication. Based on the above results, we further tested whether the cleavage of GSDMD by endogenous pS273R disrupts the GSDMD-N1–279-mediated pyroptosis and further affects ASFV replication. MA-104 cells transfected with plasmids coexpressing GSDMD and caspase-1 were chosen as positive control, which induces pyroptosis. MA-104 cells were transfected with a plasmid coexpressing GSDMD or its mutants, together with an empty vector or a plasmid expressing caspase-1 as indicated, and then infected with ASFV. Compared with the cells coexpressing GSDMD and caspase-1, ASFV replication has a slight decrease (Fig. 6E). Meanwhile, the LDH release significantly increased in the cell supernatants of the cells coexpressing GSDMD-G107A and caspase-1 (Fig. 6F). Taken together, our findings reveal that pS273R cleaves GSDMD-N1–279 to generate GSDMD-N1–107, which inhibits GSDMD-N1–279-mediated pyroptosis, resulting in the inhibition of the host antiviral innate immune responses and the enhancement of ASFV replication. The RNA virus genome can be translated into a polyprotein, which can be processed into mature functional proteins by viral protease(s) (27Fang Y. Snijder E.J. 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To test whether swine GSDMD is cleaved by the indicated viral proteases, GSDMD was coexpressed with some major proteases encoded by several viruses belonging to the Coronaviridae, Arteriviridae, and Picornaviridae. As shown in Figure 7, A–C, we found that GSDMD was cleaved to produce a 25-KD fragment by porcine epidemic diarrhea virus (PEDV) Nsp4, porcine transmissible gastroenteritis virus (TGEV) Nsp4, equine arteritis virus (EAV) Nsp4 (Fig. 7A), and by enterovirus type 71 (EV71) 3C protease (Fig. 7B), but not by foot-and-mouth disease virus (FMDV) 3C protease (Fig. 7C). These results suggest that GSDMD is also a new target of some RNA viral proteases, which may be required for these RNA viruses infection-mediated pyroptosis. The ASFV genome contains 150 to 167 ORFs encoding more than 150 viral proteins (30Dixon L. Chapman D. Netherton C. Upton C. African swine fever virus replication and genomics.Virus Res. 2013; 173: 3-14Crossref PubMed Scopus (227) Google Scholar). 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