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

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 3C^pro 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 3C^pro-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; SQSTM1; VP1; VP3; cleavage; selective autophagy.

Figure 1.

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

Figure 2.

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

Figure 3.

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

Figure 4.

SVV 3C^pro 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 3C^pro 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 3C^pro 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 3C^pro 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 3C^pro or its protease-defective mutants and SQSTM1 for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies

Figure 5.

SVV 3C^pro cleaves SQSTM1 at residues E355, Q392, and Q395. (A) 293T cells were co-transfected with plasmids encoding 3C^pro 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 3C^pro 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 3C^pro and WT SQSTM1 or SQSTM1 mutants for 24 h. The cell lysates were prepared for western blot analysis using the indicated antibodies

Figure 6.

3C^pro-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

Figure 7.

3C^pro-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