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. 2021 Mar 14;17(11):3763–3775. doi: 10.1080/15548627.2021.1897223

Selective autophagy receptor SQSTM1/ p62 inhibits Seneca Valley virus replication by targeting viral VP1 and VP3

Wei Wen a,b, Xiangmin Li a,b,c,d, Mengge Yin a,b, Haoyuan Wang a,b, Liuxin Qin a,b, Hui Li a,b, Wenqiang Liu a,b, Zekai Zhao a,b, Qiongqiong Zhao a,b, Huanchun Chen a,b,c,d, Junjie Hu e,✉, Ping Qian a,b,c,d,✉
PMCID: PMC8632295  PMID: 33719859

ABSTRACT

Macroautophagy/autophagy plays a critical role in antiviral immunity through targeting viruses and initiating host immune responses. The receptor protein, SQSTM1/p62 (sequestosome 1), plays a vital role in selective autophagy. It serves as a receptor targeting ubiquitinated proteins or pathogens to phagophores for degradation. In this study, we explored the reciprocal regulation between selective autophagy receptor SQSTM1 and Seneca Valley virus (SVV). SVV infection induced autophagy. Autophagy promoted SVV infection in pig cells but played opposite functions in human cells. Overexpression of SQSTM1 decreased viral protein production and reduced viral titers. Further study showed that SQSTM1 interacted with SVV VP1 and VP3 independent of its UBA domain. SQSTM1 targeted SVV VP1 and VP3 to phagophores for degradation to inhibit viral replication. To counteract this, SVV evolved strategies to circumvent the host autophagic machinery to promote viral replication. SVV 3Cpro targeted the receptor SQSTM1 for cleavage at glutamic acid 355, glutamine 392, and glutamine 395 and abolished its capacity to mediate selective autophagy. At the same time, the 3Cpro-mediated SQSTM1 cleavage products lost the ability to inhibit viral propagation. Collectively, our results provide evidence for selective autophagy in host against viruses and reveal potential viral strategies to evade autophagic machinery for successful pathogenesis.

Abbreviations: Baf.A1: bafilomycin A1; Co-IP: co-immunoprecipitation; hpi: h post-infection; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MOI: multiplicity of infection; PB1: N-terminal Phox/Bem1p; Rap.: rapamycin; Seneca Valley virus: SVV; SQSTM1/p62: sequestosome 1; SQSTM1-N355: residues 1 to 355 of SQSTM1; SQSTM1-C355: residues 355 to 478 of SQSTM1; SQSTM1-N392: residues 1 to 392 of SQSTM1; SQSTM1-C392: residues 392 to 478 of SQSTM1; SQSTM1-N388: residues 1 to 388 of SQSTM1; SQSTM1-N397: residues 1 to 397 of SQSTM1; UBA: ubiquitin association; Ubi: ubiquitin.

KEYWORDS: 3C protease, cleavage, SQSTM1, selective autophagy, VP1, VP3

Introduction

Seneca Valley virus (SVV) is a non-enveloped positive single-stranded virus belonging to the genus Senecavirus of family Picornaviridae [1]. SVV infection can cause severe vesicular disease which fails to distinguish with foot-and-mouth-disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS) [2–4]. Besides, SVV also exhibits potential oncolytic property against tumor cell lines of neuroendocrine origin, such as small-cell lung cancers and solid pediatric cancer cells [5,6]. The SVV genome contains a single open reading frame (ORF) that encodes a polyprotein, which is cleaved into three structural proteins and eight nonstructural proteins by 3Cpro. 3Cpro contains a conserved catalytic box with histidine (His) and cysteine (Cys) residues, which is indispensable for performing its function [7]. 3Cpro also evolves various strategies against host antiviral immunity and is essential for virus efficient replication. 3Cpro antagonized the production of host type I Interferon by targeting MAVS, TICAM1/TRIF, and TANK for cleavage and degrading DDX58/RIG-I and IRF3 through its protease activity [8–11]. It is critical to understand the interaction between host and virus, which represents potential targets for exploiting effective antiviral strategies and vaccines.

Autophagy is an intracellular catabolic process that sequesters damaged and detrimental components within double-membrane vesicles and then degrades it to maintain cellular metabolic balance and homeostasis [12,13]. While autophagy was initially thought to be nonselective, ample evidence indicates a selective autophagic degradation of cytosolic components [14]. Numerous forms of selective autophagy have been studied including degradation of ribosomes (ribophagy), intracellular bacterial, viral pathogens (xenophagy), peroxisomes (pexophagy), mitochondria (mitophagy), and ER (reticulophagy) [15]. Autophagy cargo receptors share at least one domain, the LIR domain (LC3-interacting region), allowing interaction with Atg8-family members and thus targeting the cargos to autophagosomes [16]. SQSTM1/p62 is a typical autophagy receptor that interacts with ubiquitinated substrates via its UBA domain (ubiquitin-associated domain) and that can multimerize via its PB1 domain (NH2-terminal Phox and Bem1p domain) for transferring to the autophagosome formation site. These two motifs are important for selective autophagic degradation of ubiquitinated substrates [17,18]. Besides, SQSTM1 can interact with non-ubiquitinated substrates and target it to autophagosome for autophagic degradation [19]. SQSTM1 directly bound to non-ubiquitinated Sindbis Virus capsid protein and ubiquitinated Chikungunya virus capsid and then targeted them for degradation [20,21]. The autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) targets unassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of cauliflower mosaic virus (CaMV) infection [22]. Apart from selective autophagy, SQSTM1 also plays an essential role in regulating multiple signaling pathways. SQSTM1 has a certain function on activating the NFKB pathway and type I IFN production pathway [23].

Selective autophagy plays an important role in antiviral innate immune response [24,25]. In this study, we reported that autophagy promoted or inhibited SVV propagation in a species-specific manner. Selective autophagy receptor SQSTM1 inhibited SVV propagation. SQSTM1 interacted with SVV VP1 and VP3 independent of its UBA domain and targeted them for autophagic degradation. To counteract this, SVV 3Cpro cleaved SQSTM1 at multiple distinct sites, leading to abrogate clearance of host ubiquitin conjugates and its antiviral effects. Collectively, our results provide evidence for selective autophagy into host against viruses and reveal potential viral strategies to evade autophagic machinery for successful pathogenesis.

Results

Autophagy promotes or inhibits SVV propagation in a species-specific manner

To explore whether SVV induces autophagy, the conversion level of MAP1LC3B/LC3B-I to LC3B-II was examined in SK6 cells. We found that the conversion level was significantly upregulated in SVV-infected cells compared with mock cells or UV-SVV-infected cells (Figure 1A,B). The results indicated that SVV could induce autophagy and viral replication was essential for SVV-induced autophagy. Visualizing the fluorescent puncta of LC3B was performed to further verify the result. Consistent with immunoblotting, LC3B exhibited a diffuse distribution in mock-infected cells and SVV infection leads to the accumulation of autophagosomes as detected by LC3B punctate distribution (Figure 1C). To further visualize autophagosome formation in SVV-infected cells, we utilized transmission electron microscopy (TEM) to observe it. Autophagosome-like vesicles were rarely observed in the mock-infected cells. In contrast, the number of membrane vesicles containing cytoplasmic contents increased in the cytoplasm of SVV-infected cells (Figure 1D). These results show that SVV infection can induce autophagy.

Figure 1.

Figure 1.

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Autophagy promotes SVV propagation in SK6 cells. (A and B) SK6 cells were mock-infected or infected with SVV or UV-inactivated SVV at an MOI (multiplicity of infection) of 1 for the indicated times. The expression of LC3, VP1, and ACTB was analyzed by immunoblotting with specific antibodies. (C) SK6 cells were infected with SVV (MOI = 1) for 6 h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and VP3 protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bar: 10 μm. (D) SK6 cells were mock-infected or infected with SVV for 6 h. Then the mock- and SVV-infected SK6 cells were fixed and processed for electron microscopy analysis. Black arrows indicated the structures with the characteristics of autophagosomes. (E and F) SK6 cells were incubated with rapamycin (Rap.) or DMSO for 4 h and then infected with SVV (MOI = 0.1) for the indicated times. The cell samples were analyzed by immunoblotting with specific antibodies. Virus titers were measured using a PFU assay. (G and H) shNC or shBECN1 cells were infected with SVV (MOI = 0.1) for the indicated times. The cell samples were analyzed by immunoblotting with specific antibodies. Virus titers were measured using a PFU assay

To examine the role of autophagy in SVV replication, we detected the level of viral protein VP1 and virus titers in the presence or absence of rapamycin, an inducer of autophagy. As shown in Figure 1E,F, rapamycin significantly upregulated LC3-II expression and promoted the level of VP1 expression and virus yield at 6 and 8 h post-infection (hpi) in SVV-infected SK6 cells. We further tested the effect of autophagy in SVV replication by depleting BECN1/beclin1 which is essential for autophagic flux. Consistent with the results, inhibition of autophagy decreased VP1 expression and virus propagation in SVV-infected SK6 cells (Figure 1G,H). Similar results were also observed in SVV-infected ST cells (Figure S1A,B). Surprisingly, the induction of autophagy inhibited SVV propagation in 293T cells (Figure S1C,D) and H1299 cells (Figure S1E,F), indicating that the effect of autophagy machinery on SVV replication was species-specific. Previous studies reported that SVV utilized autophagy machinery to promote proliferation in BHK cells [26]. The underlying molecular mechanism should be further studied.

SQSTM1 inhibits SVV propagation

Previous studies showed that SQSTM1 played an important role in antiviral innate immunity. To examine the role of selective autophagy receptor SQSTM1 in SVV infection, we silenced SQSTM1 expression and overexpressed SQSTM1 expression to detect its effect on SVV propagation. As shown in Figure 2A,B, SQSTM1 was significantly downregulated by specific shRNA, and knockdown of SQSTM1 enhanced viral protein VP1 expression and virus titers at 6 and 8 hpi. Consistent with the results, ectopic expression of SQSTM1 resulted in a concomitant decreased VP1 expression and virus titers (Figure 2C,D). Collectively, the results demonstrate that SQSTM1 exhibits an inhibitory effect on SVV propagation.

Figure 2.

Figure 2.

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SQSTM1 inhibits SVV propagation. (A and B) shNC or shSQSTM1 cells were infected with SVV (MOI = 0.1) for the indicated times. The cell samples were analyzed by immunoblotting for VP1 protein level and virus titers were measured using a PFU assay. (C and D) SK6 cells were transfected with plasmids encoding SQSTM1 for 20 h and then subjected to SVV infection (MOI = 0.1) for the indicated times. Cell lysates were analyzed by immunoblotting for VP1 protein level, and virus titers were measured using a PFU assay

SQSTM1 targets SVV VP1 and VP3 for degradation

Previous studies showed that SQSTM1 exhibited an antiviral effect by targeting viral capsid for autophagic degradation [20,21]. We performed co-immunoprecipitation (Co-IP) assays to analyze the interaction between structural protein (VP1, VP2, and VP3) and SQSTM1. As shown in Figure 3A, SQSTM1 was able to co-immunoprecipitate with VP1 and VP3, but not vector and VP2. Since selective autophagy receptors mainly discern the targets through its ubiquitin association (UBA) domain, we identified whether VP1 and VP3 underwent ubiquitination and then interacted with SQSTM1. Immunoprecipitation results showed that VP1 and VP3 indeed interacted with ubiquitin or ubiquitinated protein (Figure 3B). To identify whether the interaction between SQSTM1 and VP1 or VP3 was ubiquitin dependent, truncated SQSTM1 mutants losing UBA were constructed and performed for Co-IP assays. We performed Co-IP assays in shSQSTM1-293T cells to exclude that exogenous SQSTM1 lacking UBA oligomerized with endogenous SQSTM1 through PB1 domain. We found that SQSTM1∆UBA still interacted with VP1 and VP3 (Figure 3C), indicating that the interaction between SQSTM1 and VP1 or VP3 was ubiquitin independent. Since SQSTM1 induced the selective autophagic degradation of substrates by interacting with LC3, we further analyzed whether VP1 and VP3 could be present in complexes with LC3. 293T cells were co-transfected with plasmids encoding LC3 and VP1, or VP3 for 24 h and then incubated with bafilomycin A1 (Baf.A1) for 8 h, and then conducted for Co-IP assays. As shown in Figure 3D, both VP1 and VP3 precipitated with both LC3-I and LC3-II. To assess whether autophagy contributed to VP1 and VP3 degradation, SK6 cells were transfected with VP1 or VP3 for 20 h and then incubated with rapamycin for 10 h, and then incubated with Baf.A1 or not for 6 h. We observed that VP1 and VP3 were degraded in the presence of rapamycin and that the expression level of VP1 and VP3 was rescued in the presence of Baf.A1 (Figure 3F). Previous studies have shown that selective autophagy receptors target intact viruses or bacteria for autophagosome and degradation [22,27]. To further detect whether SQSTM1 targeted SVV particles, we carried out Co-IP assays and found that viral RNA failed to co-immunoprecipitate with SQSTM1 (Figure 3G), indicating that SQSTM1 did not target SVV particles. Taken together, these results reveal that SQSTM1 targets VP1 and VP3 for degradation.

Figure 3.

Figure 3.

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SQSTM1 targets SVV VP1 and VP3 for degradation. (A) 293T cells were co-transfected with plasmids encoding SQSTM1 plus the indicated viral structural protein for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. Immunoprecipitation was performed using anti-HA-tag antibodies. (B) 293T cells were co-transfected with plasmids encoding ubiquitin (Ubi) plus the indicated viral structural protein for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. Immunoprecipitation was performed using anti-Flag-tag antibodies. (C) shSQSTM1-293T cells were co-transfected with plasmids encoding VP1 or VP3 plus truncated SQSTM1 mutants for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. Immunoprecipitation was performed using anti-Flag-tag antibodies. (D) 293T cells were co-transfected with plasmids encoding LC3 plus VP1 or VP3 for 24 h and then treated with Baf.A1 (100 nM) for 8 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. Immunoprecipitation was performed using anti-HA-tag antibodies. (E) SK6 cells were transfected with plasmids encoding VP1 or VP3 for 20 h and then treated or untreated with Rap. for 10 h, and then treated or untreated with Baf.A1 for 8 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. (F) 293T cells were transfected with plasmids encoding SQSTM1 for 20 h and then mock-infected or infected with SVV (MOI = 1) for 8 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. Immunoprecipitation was performed using anti-Flag-tag antibodies. Viral RNA was detected by PCR using primers specific for SVV 3D

SVV 3Cpro targets SQSTM1 for cleavage through its protease activity

To further investigate whether SVV infection affected SQSTM1 expression, we examined the protein expression level of SQSTM1 in SK6 cells infected with SVV at different time points. Western blotting showed the decreased abundance of full-length SQSTM1, accompanied by the appearance of two close fragments, implicating virus-associated cleavage of SQSTM1 (Figure 4A). Additionally, SQSTM1 was also cleaved in SVV-infected BHK cells and 293T cells (Figure 4B,C). Although only one cleaved band was found in SVV-infected 293T cells, two cleaved bands were detected when 293T cells were co-transfected with human SQSTM1 and SVV 3Cpro (data not shown). We predicted that one of the cleavage bands was difficult to be detected in SVV-infected 293T cells. We then investigated whether 3Cpro was responsible for SQSTM1 cleavage. Cotransfection of SQSTM1 with the construct expressing viral protease 3C led to the production of two close cleavage fragments, as that observed following SVV infection. We also found that SVV 3Cpro cleaved SQSTM1 in the presence of pan-caspase inhibitor Z-VAD-FMK (Figure 4D), indicating that SQSTM1 cleavage was independent of caspase activation. In vitro cleavage assays results also showed that SQSTM1 was cleaved by purified 3Cpro in a dose-dependent manner (Figure 4E). SVV 3Cpro contained a conserved catalytic box with histidine (His) and cysteine (Cys) residues, which was essential for its protease activity. Single-site mutants H48A (3C-H48A) and C160A (3C-C160A) and the double-site mutant H48A-C160A (3C-DM) were catalytically inactive [8]. Additionally, SQSTM1 cleavage was not observed in cells co-transfected with plasmids encoding 3C-H48A, 3C-C160A, or 3C-DM (Figure 4F), indicating that the protease activity was essential for the cleavage. To explore whether 3Cpro interacted with SQSTM1, we performed Co-IP assays. As indicated in Figure 4G, SQSTM1 associated with wild-type 3Cpro and 3C-DM. Taken together, these results show that 3Cpro associates with SQSTM1 and induces SQSTM1 cleavage through its protease activity.

Figure 4.

Figure 4.

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SVV 3Cpro targets SQSTM1 for cleavage through its protease activity. (A–C) 293T, BHK, or SK6 cells were infected with SVV (MOI = 1) for the indicated times. The cell lysates were prepared for western blot analysis using the indicated antibodies. (D) 293T cells were co-transfected with plasmids encoding SQSTM1 and 3Cpro for 20 h, and then treated or untreated with Z-VAD-FMK (40 μM) for another 8 h. (E) 293T cells were co-transfected with plasmids encoding SQSTM1 and 3Cpro or 3 C-DM for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. Immunoprecipitation was performed using anti-Flag-tag antibodies. (F) 293T cells were transfected with plasmids encoding SQSTM1 for 24 h. The cell lysates were incubated with purified 3Cpro at 37 °C for 3 h and then prepared for western blot analysis using the indicated antibodies. (G) 293T cells were co-transfected with plasmids encoding WT 3Cpro or its protease-defective mutants and SQSTM1 for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies

SVV 3Cpro cleaves SQSTM1 at residues E355, Q392, and Q395

Based on the molecular weight of cleavage products, we generated the deletion mutants SQSTM1 consisting of amino acids (aa) 1 to 381 (SQSTM11–381), amino acids (aa) 1 to 407 (SQSTM11–407), and amino acids (aa) 1 to 440 (SQSTM11–440). As shown in Figure 5A, the cleavage site of the high molecular weight band was located at residues 381 to 407. Picornavirus 3C protease preferentially recognizes glutamine-glycine (Q-G) or glutamic acid-glutamine (E-Q) pairs in the viral polyprotein and host factors [28]. Based on these results, we created a series of site-directed mutations within SQSTM1 at Q or E residues, which may act as 3Cpro cleavage sites. Surprisingly, we found that only one cleavage product disappeared in SQSTM1E355A (data not shown). To detect another cleavage site, we constructed the deletion mutant SQSTM1 with N-terminal Flag tag and C-terminal His tag (Figure 5B). As shown in Figure 5C, the cleavage products were detected when SQSTM1-388 and SQSTM1-397 were in the presence of 3Cpro using an antibody against Flag tag but not using an antibody against His tag. Besides, the amount of detectable SQSTM1-397 was significantly less than that of SQSTM1-388 in the presence of 3Cpro using an antibody against his tag, suggesting that the protease cleavage occurs within the 10 amino acids. However, we still detected the cleavage product when the potential mutants were in the presence of 3Cpro (Figure 5D). We speculated that there were two or three cleavage sites within the 10 amino acids. Therefore, we constructed a series of double- or triple-site mutants and conducted a cleavage assay. As expected, the indicated cleavage product disappeared when SQSTM1Q392A,Q395A or SQSTM1E390A,Q392A,Q395A was transfected with 3Cpro (Figure 5E), demonstrating the two cleaved positions to be Q392 and Q395. To further confirm these cleaved residues, a triple mutant (SQSTM1E355A,Q392A,Q395A) was constructed and co-transfected into 293T cells with SVV 3Cpro. The results showed that SQSTM1E355A,Q392A,Q395A was resistant to 3Cpro cleavage, confirming that E355, Q392, and Q395 were recognized by SVV 3Cpro for SQSTM1 cleavage (Figure 5F).

Figure 5.

Figure 5.

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SVV 3Cpro cleaves SQSTM1 at residues E355, Q392, and Q395. (A) 293T cells were co-transfected with plasmids encoding 3Cpro and WT SQSTM1 or its truncated mutants for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. (B) schematic diagram of a series of truncated SQSTM1 mutants. (C) 293T cells were co-transfected with plasmid encoding 3Cpro and truncated SQSTM1 mutants for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies. (D–F) 293T cells were co-transfected with plasmids encoding 3Cpro and WT SQSTM1 or SQSTM1 mutants for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies

In addition, we assessed the turnover of SQSTM1, a target of autophagy that is widely used as a marker for monitoring autophagic flux. We recognized that endogenous SQSTM1 could not be used to measure flux, as it was cleaved upon SVV infection by virus-encoded 3Cpro. Here we applied a 3Cpro cleavage-resistant mutant SQSTM1 to monitor its stability. As shown in Figure S2A, the protein level of SQSTM1 mutant decreased, suggesting a complete autophagy flux. Enhanced green fluorescent protein is sensitive to lysosomal proteolysis and may diminish quickly in acidic pH. Therefore, a tandem reporter construct mCherry-GFP-LC3 was used to measure SVV infection-induced autophagy flux. As shown in Figure S2B, many LC3-positive autophagic vacuoles were yellow in cells incubated with Baf.A1. While numerous red fluorescent puncta were observed in SVV-infected cells, indicating that the formation of autophagosomes and autolysosomes was induced by SVV infection. Together, these results revealed that SVV infection induced complete autophagy.

3Cpro-mediated SQSTM1 cleavage products lose its ability to mediate selective autophagy and to inhibit SVV propagation

We further explored the functional consequences of SQSTM1 cleavage. Since the residues Q392 and Q395 were close and in the same domain, we only detected the function of cleaved fragments at Q392. Our results showed that intact SQSTM1 was capable of binding to both LC3 and polyubiquitinated proteins (Figure S3A,B). Interestingly, we observed that SQSTM1-N392 (residues 1 to 392) retained its ability to bind to LC3 (Figure S3A) but lost its association with ubiquitinated proteins (Figure S3B). As expected, the other truncated fragments of SQSTM1, which lacked both LIR and UBA domains, failed to interact with LC3 and ubiquitin chain (Figure S3A,B). Next, we assessed the subcellular localization of the truncated SQSTM1 mutants. We first examined whether truncated SQSTM1 mutants were able to form aggregates. As shown in Figure 6A,B, intact SQSTM1 formed aggregates, and truncated SQSTM1 mutants lost the ability to form aggregates. We then examined whether SQSTM1 truncations interacted with LC3 and ubiquitin. Confocal microscopy analysis revealed the punctate colocalization of transfected WT SQSTM1 with ubiquitin (70%, Figure 6C,D) and WT SQSTM1 with LC3 (80%, Figure 6E,F). However, in cells expressing SQSTM1 truncations, the colocalization ratio was significantly decreased (Figure 6C–F). These results indicated that the association between SQSTM1 truncations with LC3 and ubiquitin within the punctate structures was disrupted. Self-oligomerization of SQSTM1 is critical for its localization to the autophagosome formation site [29]. Although SQSTM1-N392 retained its ability of binding to LC3, it lost the ability to form aggregates and to interact with ubiquitin. So it could not mediate selective autophagy.

Figure 6.

Figure 6.

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3Cpro-mediated SQSTM1 cleavage products lose their ability to mediate selective autophagy. (A and B) SK6 cells were transfected with plasmids encoding WT SQSTM1 or truncated SQSTM1 mutants for 24 h. The cells were then fixed and processed for indirect immunofluorescence using anti-Flag-tag antibodies, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bar: 10 μm. The percentage of cells with punctate SQSTM1 was calculated in three independent experiments. At least 40 cells were counted each time. Data are represented as means ±SD. Student’s t-test: *P < 0.05, ** P < 0.01, ***P < 0.001, ns = not significant. (C–F) SK6 cells were co-transfected with plasmids encoding WT SQSTM1 or truncated SQSTM1 mutants and Ubi or LC3 for 24 h. The cells were then fixed and processed for indirect immunofluorescence using anti-Flag-tag antibodies, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bar: 10 μm. The percentage of cells with punctate colocalization was calculated in three independent experiments. At least 40 cells were counted each time. Data are represented as means ±SD. Student’s t-test: *P < 0.05, ** P < 0.01, ***P < 0.001, ns = not significant

Next, we detected the binding affinity to VP1 or VP3 for 3Cpro-mediated SQSTM1 cleavage products. The cleavage products lost the ability to interact with VP1, but SQSTM1-N355 and SQSTM1-N392 still contained a certain ability to interact with VP3 (Figure 7A,B). To assess whether 3Cpro-mediated SQSTM1 cleavage abolished its antiviral activity, individual deletion mutants were transiently transfected into SK6 cells followed by SVV infection for 8 h. As shown in Figure 7C,D, compared with WT SQSTM1, the inhibitory effects of the deletion mutants against SVV were significantly decreased. Taken together, 3Cpro-mediated SQSTM1 cleavage products lose their ability to mediate selective autophagy and to inhibit SVV replication.

Figure 7.

Figure 7.

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3Cpro-mediated SQSTM1 cleavage products lose their ability to inhibit SVV propagation. (A and B) SQSTM1 cells were transfected with plasmids encoding WT SQSTM1 or truncated SQSTM1 mutants for 20 h and then infected with SVV (MOI = 0.1) for 8 h. The cell samples were analyzed by immunoblotting for VP1 protein level and virus titers were measured using a PFU assay

Discussion

Autophagy regulates host immunity and mediates the selective degradation of viral components, but several viruses have evolved mechanisms by which autophagic processes are counteracted to promote virus replication [30,31]. Here, we demonstrate that autophagy promotes SVV infection in pig cells. Yet, in human cultured cells, SVV triggers an autophagic response that inhibits viral replication. SQSTM1-mediated selective autophagy targets VP1 and VP3 for degradation to inhibit SVV replication. Furthermore, SVV 3Cpro cleaves SQSTM1 to abolish its ability to mediate selective autophagy and to inhibit SVV replication. Our results provide evidence for selective autophagy in host against viruses and reveal potential viral strategies to evade autophagic machinery for successful pathogenesis.

Selective autophagy plays an important role in the clearance of protein aggregates, damaged mitochondria, bacteria, and viruses. Previous studies have reported that SQSTM1 targets ubiquitin-dependent and -independent virus capsids for autophagic clearance, including herpes simplex virus (HSV-1), foot-and-mouth disease virus (FMDV), chikungunya virus (CHIKV), and sindbis virus (SINV) [20,21,32,33]. In this study, we found that VP1 and VP3 interacted with SQSTM1. Though VP1 and VP3 were subjected to ubiquitination, SQSTM1 interacted with VP1 and VP3 independent of its UBA domain. SQSTM1 interacts with LC3 through its LIR domain, a critical process required for SQSTM1 recruitment into the phagophore [14,34]. Our results showed that VP1 and VP3 interacted with LC3, which provided a basis for autophagy to degrade VP1 and VP3. Recent studies have indicated a role for SQSTM1 in targeting ubiquitin-coated Salmonella for autophagic degradation [35]. NBR1-mediated xenophagy of viral capsids and viral particles inhibits cauliflower mosaic virus (CaMV) infection [22]. To analyze whether SQSTM1 directly targets viral particles, we carried out RNA binding protein immunoprecipitation. We found that SQSTM1 failed to coimmunoprecipitate with viral RNA, indicating that SQSTM1 did not target SVV particles.

Viruses have evolved various strategies to counteract host antiviral response. CaMV infection induces the formation of inclusion bodies to protect virus particles from NBR1-mediated degradation [22]. Coxsackievirus B3 (CVB3) 2A protease cleaves DAP5 to facilitate viral replication and enhance apoptosis by altering the translation of IRES-containing genes [36]. In the present study, SQSTM1 played a potential role in virophagy by interacting with VP1 and VP3. Interestingly, SQSTM1 was cleaved during SVV infection by viral 3Cpro at multiple distinct sites (E355A, Q392A, and Q395A), mediated by its protease activity. Since the residues Q392 and Q395 are very close and in the same domain, we only detect two 3Cpro-mediated SQSTM1 cleavage bands. In addition to SVV, cleavage of SQSTM1 at G241 (the glycine residue at position 241) has been confirmed on CVB3, poliovirus, and enterovirus D78 infection by viral 2Apro [37–40], suggesting a common picornavirus strategy to counteract selective autophagy receptor. To our knowledge, our study was also the first to show that SQSTM1 cleavage by viral 3Cpro accured at multiple residues to abrogate its function of mediating selective autophagy. 2Apro-mediated cleavage of SQSTM1 occurs within its respective TRAF6-binding domain. The C-terminal of SQSTM1, which contains LIR domain, still interacted with LC3. SVV 3Cpro-mediated cleavage site E355 occurred within its respective LC3-interacting region, leading to disrupt SQSTM1-LC3 interaction, but SQSTM1-N392 still retained the ability to interact with LC3. Consistent with SVV 3Cpro-mediated SQSTM1 cleavage products, 2A-mediated cleavage SQSTM1 fragments lost its ability to bind ubiquitin and to form punctate. Previous studies showed that SQSTM1 targeting the autophagosome formation site requires self-oligomerization [29]. Only VP3 was still capable of binding to SQSTM1-N355 and SQSTM1-N392, but the degree of interaction was significantly lower than that of wild-type SQSTM1. So all cleavage products could not target VP1 or VP3 to autophagosome and lost antiviral activity. Due to 3Cpro cleaving SQSTM1, endogenous SQSTM1 was not a reliable marker to evaluate autophagic flux in the context of SVV infection. Therefore, we used a 3Cpro non-cleavable SQSTM1 and tandem mCherry-GFP-LC3 reporter construct to examine autophagic flux. We demonstrate that SVV infection results in a significantly complete autophagic flux. The current study addressed the limitations of previous research, in which SQSTM1 expression was used to evaluate whether a complete autophagic process was induced in SVV-infected cells [26]. CVB3 and EV-D68 infection inhibited autophagic flux via disruption of the SNARE complex to facilitate their propagation. SNAP29 was cleaved by viral 3Cpro at Q161. STX17 expression was increased in EV-D68-infected cells but decreased in CVB3-infected cells [37,40]. However, EV71 and FMDV infection induced a complete autophagic flux. The formation of autolysosomes during EV71 infection was important for virus replication. And non-structural protein 2BC interacted with STX17 and SNAP29 [41–43], which contributed to virus replication and may promote autophagic degradation. These indicated that picornavirus adopted different approaches to manipulate autophagic machinery to promote viral replication.

Autophagy, a highly conserved cellular degradative pathway, serves as an antiviral defense mechanism or as a pro-viral process during virus infection [31]. In our study, autophagy limited SVV replication in human cells, nevertheless, it promoted SVV propagation in pig cells. Previous studies reported that the absence of NDP52-nsP2 interaction in mouse cells might account for the lower permissiveness of mice to CHIKV relative to humans [44]. As the regulation of autophagy to virus replication is a complicated process, the molecular mechanisms underlying these species-specific differences should be further studied.

Taken together, our study provides a novel insight into the mechanisms by which SVV evades host virophagy through 3Cpro-mediated cleavage of SQSTM1 to promote viral replication. Our findings can be applied for antiviral strategies development against SVV infection and pathogenesis.

Materials and methods

Cell culture, transfection, and drug treatment

Human embryonic kidney 293T cells (HEK293T cells; ATCC, CRL-11268), ST cells (swine testis; ATCC, CRL1746), and baby hamster kidney cells (BHK21 cells; ATCC, CCL-10) were cultured in Dulbecco’s modified essential medium (HyClone, SH30022.01) containing 10% fetal bovine serum (Gibco, 16000044) and 100 U/ml of penicillin (GENVIEW, GA3502) at 37 °C in a humidified 5% CO2 incubator. Swine kidney cells (SK6 cells; Wuhan University) were grown in Minimum Essential Medium (Gibco, 8119027) containing 10% FBS, 100 U/ml of penicillin grown under the same conditions as those described above. NCI-H1299 (human non-small cell lung cancer; ATCC, CRL-5803) were grown in RPMI 1640 medium containing 10% FBS, 100 U/ml of penicillin grown under the same conditions as those described above. For transfection, the cells were transiently transfected with plasmids using Lipofectamine 2000 (Invitrogen, 11668019) following the manufacturer’s protocols.

For caspase inhibition experiments, Dimethyl sulfoxide (Beyotime Biotechnology, ST038) or caspase inhibitor Z-VAD-FMK (Beyotime Biotechnology, C1202) was added to the medium.

Viruses, plasmid, and antibodies

The SVV strain HB-CH-2016 (GenBank accession number KX377924) used in this study was isolated from piglets with PIVD [45]. SVV was propagated and virus titers were determined using a plaque-forming unit (PFU) assay in BHK21 cells.

SVV 3Cpro was cloned into vector pCAGGS-HA (miaolingbio, P0166). SQSTM1 were amplified from SK6 cells cDNA and then cloned into vector pCDNA3.1-3Flag (miaolingbio, P0157). SQSTM mutants were cloned into pCDNA3.1-3Flag. Mutagenesis of SVV 3Cpro was generated using overlap PCR and cloned into vector pCAGGS-HA. GFP-LC3 and mCherry-GFP-LC3 were kindly provided by Hongbo Zhou (Huazhong Agricultural University).

Mouse monoclonal antibodies against HA tag (M180-3) and rabbit polyclonal antibodies against Flag tag (M385-3L) were purchased from Medical and Biological Laboratories (MBL). Mouse monoclonal antibodies against Flag tag (66008-3-Ig) were purchased from Proteintech Group. Alexa Fluor 555 goat anti-mouse (A32727) or -rabbit (A32732) antibodies and Alexa Fluor 488 goat anti-rabbit (A32731) or -mouse (A32723) antibodies were obtained from Invitrogen. Mouse anti-GAPDH (60004-1-Ig), anti-ACTB/β-actin (60008-1-Ig), and anti-TUBA1B/α-tubulin (66031-1-Ig) monoclonal antibodies were obtained from Proteintech Group. Rabbit polyclonal antibodies against SQSTM1 (A11483), BECN1 (A17028), and LC3B (A7198) were obtained from ABclonal. Anti-SVV VP1 polyclonal antibodies and anti-SVV VP3 monoclonal antibodies were prepared in our laboratory.

Lentivirus packaging

The shRNA constructs were designed by using the pLKO.1 vector (miaolingbio, P0258) in accordance with the manufacturer’s instructions. 293T cells were co-transfected with 0.2 μg pCMV-VSVg (miaolingbio, P0269), 0.8 μg pCMV-Gag/pol (miaolingbio, P0447), and 1 μg pLKO.1 for 48 h to generate lentivirus [46]. The medium was harvested and centrifuged at 12,000 g for 5 min and then divided and stored at −80 °C. When the lentivirus was used for infection, SK6 cells were seeded in 12-well plates for 24 h, and then lentivirus containing 7 μg/ml polybrene (Beyotime Biotechnology, C0351) was added. After 12 h incubation, the medium was replaced with fresh complete growth media for another 36 h.

Western blot and Co-IP analysis

Cells were harvested and treated with lysis buffer (1.19% HEPES, 0.88% NaCl, 0.04% EDTA, 1% NP-40 [Beyotime Biotechnology, P0013F]) containing a protease inhibitor (bimake, B14011), and then incubated on ice for 30 min. Equal amounts of protein were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then the separated protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, 46978100). For the Co-IP assay, cell lysates were incubated with anti-HA-tag or anti-Flag-tag monoclonal antibodies in a rolling incubator at 4 °C overnight. Then the mixture was incubated with Protein A + G Agarose (Beyotime Biotechnology, P2028) at 4 °C for 6 h. Subsequently, the complex was washed five times with cold lysis buffer and then an immunoblotting analysis was conducted.

In vitro cleavage assay

To obtain SVV 3Cpro, E. coli BL21 (DE3) cells harboring pET28a-6×His-3Cpro were grown in LB medium supplemented with 50 µg/ml kanamycin. Protein expression was induced overnight at 16 with 0.5 mM isopropyl-B-D-thiogalactopyranoside (Beyotime Biotechnology, ST098-5g) after OD600 nm reached 0.8. Cells were harvested and lysed by sonication on ice in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 5 mM imidazole). After centrifugation at 12,000 g at 4°C for 1 h, the supernatant was collected and filtered. The supernatant was run through a Ni-NTA column (GE Healthcare, 10291457) at a flow rate of 0.1 ml/min, followed by extensive washing with lysis buffer. The recombinant SVV 3Cpro eluates were dialyzed three times against phosphate-buffered saline (PBS, pH 7.2; Beyotime Biotechnology, C0221A) at 4°C to remove imidazole. Fifty µg 293T cell extracts expressing SQSTM1 were incubated with increasing dose 3Cpro at 37 °C for the indicated time. The reaction was stopped by addition of SDS-PAGE sample buffer. Protein cleavage was analyzed by western blotting.

Immunofluorescence analysis

Cells were fixed with 4% paraformaldehyde for 30 min and permeated with 0.2% Triton X-100 (Beyotime Biotechnology, P0096) at room temperature for 20 min, and then blocked with 3% bovine serum albumin (BSA; GENVIEW, FA016) in PBS for 1 h. Subsequently, the primary antibodies were diluted in PBS at the indicated concentration at 37 °C for 2 h. After washed five times with PBS, the cells were incubated with secondary antibodies at room temperature for 1 h. Fluorescence images were acquired with a confocal laser scanning microscope (Carl Zeiss, LSM 510 Meta).

Statistical analysis

Statistical analysis was conducted using GraphPad Prism software, version 5. All results are determined at least three times of independent experiments. The various treatments were compared using an unpaired, two-tailed Student t-test with an assumption of unequal variance. P-value < 0.05 was considered statistically significant.

Supplementary Material

Supplemental Material
Click here for additional data file. (9.2MB, zip)

Funding Statement

This work was supported by grants from the National Program on Key Research Project of China [2018YFD0500204]; the National Natural Science Foundation of China [31772749 and 32072841]; the Fundamental Research Funds for the Central Universities [2662017PY108]; and Natural Science Foundation of Hubei Province [2019CFA010].

Disclosure statement

The authors have no financial conflicts of interest.

Supplementary material

Supplemental data for this article can be accessed here.

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Supplementary Materials

Supplemental Material
Click here for additional data file. (9.2MB, zip)

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