Fig 1.
SVA infection leads to lysophagy.
(A-E) After SVA infection, BHK-21 cells were collected at different time points, and the expression levels of lysophagy related proteins and SVA VP2 proteins were examined. (F-J) The cytoplasm fraction of BHK-21 cells were extracted at different time points after SVA infection, and the protein levels of CTSD and CTSB (mature and precursor) in the two fractions were detected by Western Blot.(K) Comparison between lysophagy and LLOME (1 mM) addition in BHK-21 cells and PK-15 cells infected by SVA under Transmission electron microscope. In the enlarged images, the infected and LLOME cells present a damaged lysosome with a single-layer membrane structure typical of autophagosomes compared to the control group (red arrow). Scale bar:5 μm,500 nm.(L) Cell sections from SVA infected and control groups were incubated with primary anti-Gal3 antibody.After incubation with secondary antibodies conjugated to 5nm colloidal gold particles, representative TEM images were observed and photographed to record, and the gold particles were mainly localized in the interior of the autophagic vesicles. Scale bar: 500 nm (M,N) Colocalization of LAMP1 with Gal3 in cells with or without SVA infection(6 hpi), five single-cell fields of view were randomly selected from each group, and the co-localization coefficients (Pearson coefficients) of the two channels were statistically analyzed. Scale bar: 20 μm. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
Fig 2.
Protective effect of DLC on lysosomes after SVA infection.
(A,B) Effect of DLC and SVA infection on the expression of LAMP1 protein. (C,D) LAMP1 protein levels were detected after treatment with the protein synthesis inhibitor CHX (5 μM) for 0, 3, 6, and 12 h within 12 hours of SVA infection. (E,F) After viral infection, CHX (5 μM) was used separately or in combination to determine the pathway by which DLC maintains LAMP1 protein. (G) The mRNA expression of LAMP1 in cells treated with DLC (5 μM) at different time points after infection. (H,I) Cells were incubated with Lysosensor Green DND-189(0.1 μM)for 1h, and the lysosome fluorescence intensity was detected under a laser confocal microscope. Three fields were selected and the average fluorescence intensity was statistically analyzed. (J,K) The lysosomal membrane permeability(LMP) was detected by acridine orange staining, and the red fluorescence intensity (Excitation wavelength 555nm, emission wavelength 617nm) in any three fields was counted and statistically analyzed. (L-O) BHK-21 cells were pretreated with DMSO, LLOME (1mM), NH4Cl (2 μM), over-expressed HA-LAMP1 and LAMP1 siRNA, and then infected with SVA at an MOI of 0.01 for 24h. The cell lysate was collected. The protein expression of LAMP1 and VP2 was detected. Scale bar: 10 μm, 20 μm. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
Fig 3.
DLC reduced the autophagic flux that was elevated by SVA infection without blocking autophagy.
(A-C) The expression levels of VP2 and LC3B proteins (D,E) GFP-mCherry-LC3B plasmid was transfected into BHK-21 cells 12 h later, the transfected cells were infected with the autophagy inhibitor Baf A1(5 μM) and the autophagy activator Rapamycin (5 μM) and DLC (5 μM) respectively or in combination. The autophagy flow was detected by confocal microscopy.The ratio of mCherry spots to mCherry+GFP spots in at least 10 individual cells was analyzed and statistically analyzed (n > 10). (F-H) The cells were infected with SVA at a MOI of 0.01 and treated with different concentrations of DLC (0,2,5 μm) for 24 hpi. The cell lysates were collected to detect the protein expressions of ULK1, ATG16L and Beclin1 in the autophagy pathway. Scale bar: 20 μm. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
Fig 4.
DLC reduces ubiquitin-proteasome degradation of LAMP1 induced by SVA infection and inhibits lysophagy.
(A) The ubiquitination level of LAMP1 treated with CQ (10 μM) and MG132 (50 nM) before and after SVA infection. (B,C) Effect of CQ (50 μM) and MG132 (50 nM) on LAMP1 protein levels before and after SVA infection. (D,E) Effects of DLC alone or in combination with MG132 (50 nM) on LAMP1 protein levels after SVA infection. (F) Effect of DLC on the interaction between endogenous TRIM16 and Gal3 in SVA infected cells by Co-IP (After Gal protein was immunoprecipitated, Gal3 and TRIM16 proteins were respectively used for detection). (G,H) Colocalization changes of TRIM16 and Gal3 in cells with and without DLC (5 μM) after SVA infection (0.01MOI, 24 hpi) and quantitative analysis.(*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
Fig 5.
DLC works by binding endogenous sulfur dioxide.
(A, B) After 0 and 24 h of SVA infection, probe CIJ (5 μM) for the detection of endogenous sulfur dioxide was added, Images were obtained at 420-600 nm for blue fluorescence and 600-700 nm for red fluorescence, respectively, and the relative fluorescence intensity ratios of blue/red for individual cells in three random fields were selected and counted. (C, D) Changes in colocalization of LAMP1 and Gal3 cells in response to SVA infection (0.01MIOI, 12hpi) and DLC (5 μM) treatment were examined by confocal microscopy after inhibition of endogenous SO2 production, Through statistical analysis of the Pearson’s coefficients of the co-localization of the two in three random fields of view.Scale bar: 20 μm. (E,F) The number of autophagosomes in the above groups was observed by electron microscopy, and the number of autophagosomes in 10 randomly selected cells was counted and counted, Scale bar:5 μm (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
Fig 6.
DLC directly binds to LAMP1 and regulates the sulfenylation of LAMP1 through SO2 to reduce the production of SVA.
(A) Predicted structure of LAMP1(blue) and DLC (yellow). The binding sites of DLC on LAMP1 protein were predicted by Docking (Glu140, His162, Met163, Asn164, Asn322, Ser324, Ile325, Tyr336, Val346, Thr347, Lys348, Cys375, Glu374) (B) The molecular binding assay through MST experiments (Signal to Noise Ratio>4). (C) The sulfenylation labeling of proteins in cells and the flow diagram of immunoblotting.(D,E) After different treatments of BHK-21 cells, cells were harvested according to the procedure shown in (D-E), the thiolation level of LAMP1 protein was measured, and the reversibility of this oxidation was confirmed by DTT (10mM). (F,G) The sulfonylation of LAMP1 by DLC was detected by adding SO2 donor NaHSO3 (500 nM); - (H,I) Sulfenylation level of Cys375A-LAMP1 after SVA infection and DLC treatment. (J,K) The expression of VP2 in SVA infected BHK-21 cells treated with different concentrations of SO2 donor NaHSO3. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
Fig 7.
DLC reduces the production of SVA virus in BHK-21 and PK-15 cells.
(A-C) BHK-21 cells and PK-15 cells were pretreated with 0,2,5,10 μM DLC for 30 min before virus infection, and the virus venom was added according to 0.01 MOI. Protein samples were collected and the expression level of virus VP2 in the cells was detected by western blot assay. (D,E) The effects of DLC on SVA virus titer in BHK-21 cells and PK-15 cells were detected by TCID50 method. (F) Immunofluorescence assay was used to observe the number of VP2-positive cells in BHK-21 and PK-15 cells after SVA infection and DLC treatment. Scale bar: 50 μm (G-I) After transfection of Vec and HA-C375A-LAMP1 in BHK-21 cells, DLC was added to detect the protein levels of LAMP1 and VP2. (J-L)After transfection of Vec and HA-C375A-LAMP1 in BHK-21 cells, the colocalization changes of Gal3 and LAMP1 were detected under confocal microscopy.Scale bar: 20 μm. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
Fig 8.
DLC reduced the proliferation of SVA on mice and the damage to organs.
(A) Experimental procedure of SVA injection and DLC injection in mice. (B) Determination of SVA viral load (Log10 RNA Copies/ mg)in various organs of mice in different groups by qPCR. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 5) (C) 21-day average daily gain of mice in different groups. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 5). (D) Pathological changes of various organs after virus infection and DLC treatment observed by HE staining.
Fig 9.
Schematic diagram of DLC promoting sulfinylation of LAMP1 and inhibiting lysophagy induced by SVA infection.
In the absence of DLC, SVA infection caused lysosomal damage, increased LAMP1 protein ubiquitination and decreased SO2 levels. In the presence of DLC, the SO2 level is increased, and DLC binds it and regulates its function. By binding to LAMP1, DLC increases the sulfation of 375 cysteine and reduces lysosomal damage caused by SVA infection. Lysophagy is inhibited, thereby inhibited lysosomal damage.