CN115998876B - Application of SAC1 as hepatitis B treatment target - Google Patents

Abstract

The invention discloses application of a substance targeting SAC1 in preparing a medicament for treating hepatitis B, wherein the medicament enables SAC1 to be over-expressed; also discloses the application of SAC1 protein or recombinant plasmid expressing SAC1 protein in preparing medicine for treating hepatitis B. The research of the invention shows that SAC1 can obviously inhibit HBV replication by promoting autophagy degradation of HBV virions, and SAC1 has good application prospect as a new potential target point for anti-HBV treatment. The study of the invention shows that SAC1 deletion causes accumulation of a large amount of PI4P in Golgi-ATG 9 vesicle-autophagosome, and the autophagy-lysosome fusion is blocked by inhibiting interaction between SNAP29 and VAMP8, so that autophagy degradation of HBV virions is reduced, HBV replication is enhanced, and a novel anti-HBV strategy is provided from the perspective of phospholipid metabolism.

Description

Application of SAC1 as hepatitis B treatment target

Technical Field

The invention relates to the technical field of biomedicine and medicine, in particular to application of SAC1 as a hepatitis B treatment target.

Background

The risk of cirrhosis and even hepatocellular carcinoma in patients infected with chronic Hepatitis B Virus (HBV) is high, and there is growing evidence that HBV production is closely related to intracellular lipid metabolism. Phosphatidylinositol lipids play an important role in lipid signaling, membrane recognition, carrier transport, and viral replication. However, there is currently no relevant document as to whether phosphatidylinositol can regulate HBV replication.

SAC1 phosphatidylinositol phosphatase (SACM 1L/SAC 1) is a key lipid phosphatase for phosphatidylinositol-4-phosphate (PtdIns 4P/PI 4P), an intact membrane protein, present in the Endoplasmic Reticulum (ER) and in the Golgi apparatus, circulating between the endoplasmic reticulum and the early Golgi apparatus. Studies have shown that SAC1 promotes replication of certain viruses, including Hepatitis C Virus (HCV) and epstein barr virus, in vitro by recruiting large amounts of PI4P to the viral replication site. Popescu et al reported that in Huh7 cells with SAC1 Knockdown (KO), SAC1 deletion resulted in PI4P accumulation, which prevented HBV envelope protein transport to the polysomes (MVBs), thus inhibiting HBV viral nucleocapsid packaging and secretion, suggesting that SAC1 may be a key host cytokine regulating viral assembly and secretion. So far, it is not clear whether SAC1 regulates other steps in HBV life cycle.

Disclosure of Invention

Phosphatidylinositol lipids play an important role in lipid signaling, membrane recognition, carrier transport, and viral replication. Previous studies have shown that SAC1 phosphatidylinositol phosphatase (SACM 1L/SAC 1) with phosphatidylinositol-4-phosphate (PI 4P) as a substrate greatly affects replication of several viruses in vitro. However, whether and how SAC1 regulates replication of Hepatitis B Virus (HBV) is unclear. In the studies of the present invention, we found that SAC1 silencing significantly increased HBV replication and production of hepatitis b virus surface antigen, whereas SAC1 overexpression produced the opposite effect.

Based on this, the present application provides the use of a substance targeting SAC1 for the preparation of a medicament for the treatment of hepatitis b, which medicament overexpresses SAC 1.

In the technical scheme, the medicine is a SAC1 promoter.

Over-expressed SAC1 inhibits HBV replication.

SAC1 prevents replication of hepatitis b virus by enhancing autophagy degradation mediated by autophagosome-lysosomal fusion.

SAC1 enhances autophagosome-lysosomal fusion by promoting interactions between synaptosome associated protein 29 (SNAP 29) and vesicle associated membrane protein 8 (VAMP 8).

The invention also provides application of the SAC1 protein or the recombinant plasmid expressing the SAC1 protein in preparing medicaments for treating hepatitis B.

The recombinant plasmid for expressing the SAC1 protein is obtained by accessing a gene for encoding the SAC1 protein into an expression vector.

In the technical scheme, the recombinant plasmid is a SAC1-myc plasmid.

In the above technical solution, the drug includes a pharmaceutically acceptable carrier.

In the technical scheme, the medicine is tablets, capsules, injections, granules, powder, pills or oral liquid.

The beneficial effects of the invention are as follows:

the present inventors have found that SAC1 silencing significantly increases HBV replication and production of hepatitis b virus surface antigen, whereas SAC1 overexpression produces the opposite effect. SAC1 overexpression inhibited HBV replication in a hydrodynamic injection model of chronically HBV infected mice. Through further mechanistic studies, SAC1 silencing was found to increase the number of autophagosomes containing HBV and PI4P levels on autophagosome membranes, SAC1 silencing prevented autophagosome-lysosomal fusion by inhibiting the interaction between synaptosome associated protein 29 (SNAP 29) and vesicle associated membrane protein 8 (VAMP 8). The research of the invention shows that SAC1 can obviously inhibit HBV replication by promoting autophagy degradation of HBV virions, and SAC1 has good application prospect as a new potential target point for anti-HBV treatment.

The study of the invention shows that SAC1 deletion causes accumulation of a large amount of PI4P in Golgi-ATG 9 vesicle-autophagosome, and the interaction between SNAP29 and VAMP8 is inhibited to block autophagy-lysosome fusion, so that autophagy degradation of HBV virions is reduced, and HBV replication is enhanced. The research result of the invention provides a new anti-HBV strategy from the perspective of phospholipid metabolism.

Drawings

FIG. 1 is a graph showing the results of SAC1 silencing experiments, wherein FIGS. A-B are graphs showing the secretion of (A) HBsAg and the content of (B) HBsAg in cell lysates in culture supernatants by ELISA method after transfection of 40nM of both SAC 1-specific siRNA (siSAC 1) or control siRNA (siNC) into HepG2.2.15 cells, co-transfection of pHBV1.3 plasmid and 40nMsiSAC1 or siNC into Huh7 cells for 72 h; (C) 48 hours after transfection, intracellular HBsAg was stained and finally imaged with confocal microscopy (scale bar, 25 μm) and fluorescence intensity of HBsAg was analyzed using imageJ software; (D) Analyzing the expression of SAC1 and HBcAg by western blotting, and taking beta-actin as a control; (E-F) detecting HBVDNA levels by real-time qPCR (E) and Southernblotting (F), respectively; the scale bar is 25 μm. * p <0.05; * P <0.01; ns is not significant.

FIG. 2 is an experiment showing the effect of AC1 overexpression on HBV replication and viral gene expression, wherein panels A-B show the levels of HBsAg in culture supernatants (A) and cell lysates (B) in Huh7 cells, after 72h of co-transfection of pHBV1.3 plasmid with SAC1-myc or empty vector pcDNA3.1; (C) In Huh7 cells, the pHBV1.3 plasmid and GFP-SAC1 or control vector pEGFP-C1 were co-transfected, and after 48 hours of transfection, the cells were HBsAg stained and finally imaged with confocal microscopy (scale bar, 25 μm); the fluorescence intensity of hbsag was analyzed with ImageJ software; (D) Detecting the expression level of SAC1 and HBcAg by western blotting; (E-F) detecting the level of HBV DNA by real-time qPCR and southern blotting, respectively; * p <0.05; * P <0.01; ns is not significant.

FIG. 3 is the results of SAC1 overexpression experiments in a mouse model of chronic HBV infection, wherein (A) male C57BL/6 mice were injected with plasmid pAAV-HBV1.2 and Sac1-myc or empty vector pcDNA3 by high pressure tail vein; (B) Mouse serum samples were taken 4, 7, 14, 21, 28, 35, 42 days after injection. ELISA method for detecting serological HBsAg at each time point; (C) Liver tissues were collected on day 14 and day 42 post-injection, two samples from each group were taken for immunohistochemical detection, liver tissue sections were incubated with rabbit anti-HBc antibody and goat anti-rabbit IgG secondary antibody, and HBcAg positive hepatocytes were counted (200 fold magnification); (D-E) isolating and detecting HBVDNA levels in mouse liver by real-time qpcr (D) and Southernblotting (E), respectively; * p <0.05; * P <0.01; ns is not significant.

FIG. 4 is the experimental results of the effect of SAC1 silencing and overexpression on PI4P levels in liver cancer cell lines, co-transfected pHBV1.3 plasmid and 40nM of both SAC 1-specific siRNA (siSAC 1) or control siRNA (siNC) in Huh7 cells; (A) 48h after transfection, cells were fixed, incubated with horse anti-HBsAg antibody, rabbit anti-SAC 1 antibody and mouse anti-PI 4P antibody, and finally, cells were imaged with confocal microscope (scale bar, 25 μm), and fluorescence intensity of PI4P was analyzed with imageJ software; (B) Huh7 cells were transfected with GFP-SAC1 or empty vector pEGFP-C1, 48h after transfection, cell fixation, incubation with equine anti-HBsAg antibody and mouse anti-PI 4P antibody, and staining with AlexaFluor594 conjugated anti-equine IgG antibody; (C-D) harvesting cells 72h after transfection, and measuring the content of PI4P in the cell lysate by ELISA; (E-G) cells were fixed 48h after transfection and incubated with horse anti-HBsAg, mouse anti-PI 4P, rabbit anti-GM 130 (E), ATG9 (F), LC3 (G) antibodies; percentage co-localization of PI4P with GM130, ATG9 or LC3 was calculated by confocal microscopy imaging (scale bar, 25 μm); * p <0.05; * P <0.01; ns is not significant.

FIG. 5 is the experimental results of the effect of SAC1 silencing on autophagy flux and HBV replication: (A-B) HepG2.2.15 cells were transfected with 40nM of both SAC 1-specific siRNA (siSAC 1) or control siRNA (siNC), then treated with 5. Mu. MVPS34-IN1 (A) or 2. Mu. MTorin-1 (B) and control DMSO for 48h, and the levels of secreted HBsAg IN the culture supernatant and HBsAg IN the cell lysate were detected by ELISA, and wild-type (WT) Huh7 cells and ATG5KO Huh7 cells were co-transfected with plasmids pHBV1.3 and siSAC1 or SAC1-myc, respectively; 72h after (C-D) transfection, the western blot detects the expression levels of SAC1, ATG5 and HBc; (E-F) ELISA to detect the secretion level of HBsAg in culture supernatant and cell lysate; * p <0.05, < p <0.01; ns is not significant.

FIG. 6 is experimental results of the effect of SAC1 silencing or overexpression on autophagosome number and autophagy degradation in HBV transfected Huh7 cells: (A-B) cotransfection of pHBV1.3 plasmid and 40nMsiRNA (siSAC 1) or control siRNA (siNC), (A) cell fixation, incubation with horse anti-HBs antibody, rabbit anti-SAC 1 antibody and mouse anti-LC 3 antibody, followed by staining with AlexaFluor 488-conjugated mouse anti-, alexaFluor 594-conjugated horse anti and AlexaFluor 647-conjugated rabbit anti-IgG antibody; (B) The expression of LC3 and p62 in the cell lysate was analyzed 72h after transfection by western blotting; (C-D) Co-transfection of Huh7 cells with pHBV1.3 plasmid and GFP-SAC1 or empty vector pEGFP-C1: (C) After 48h of transfection, the transfected cells were fixed, incubated with horse anti-HBs and rabbit anti-LC 3 antibodies, then stained with AlexaFluor594 conjugated rabbit anti-and AlexaFluor647 conjugated horse anti-IgG antibodies, imaged with confocal microscopy, counted LC3 spots for each cell and statistically analyzed, scale bar 25 μm; (D) The cell lysates were analyzed for LC3 and p62 tables 72h after transfection using western blotting; (E) Co-transfecting Huh7 cells with mCherry-GFP-LC3 plasmid and 40nMsiSAC1 or siNC; cells treated with 10 μm Chloroquine (CQ) for 24h served as positive control; punctiform fluorescence intensities of mCherry-LC3 and GFP-LC3 were shown by confocal microscopy imaging; a scale; 25 μm; * p <0.05, < p <0.01; ns is not significant.

FIG. 7 is an experiment showing the effect of SAC1 silencing on autophagy degradability, huh7 cells co-transfected with pHBV1.3 plasmid and 40nMsiRNA (siSAC 1) or control siRNA (siNC): (A) After 48h of transfection, the eggs were incubated with 10. Mu.g/ml DQ-BSA for 30min and the accumulated fluorescent signal of DQ-BSA was analyzed by confocal microscopy, with a scale of 25. Mu.m; (B) After 48 hours of transfection, cells were stained with 100nMLysoTrackerRed for 1 hour, and the fluorescence intensity of LysoTrackerRed was analyzed with a confocal microscope, with a scale of 25. Mu.m; (C) Co-transfecting Huh7 cells with GFP-LC3 plasmid and 40nMsiSAC1 or siNC, and fixing the cells 48h after transfection, confocal microscopy observing co-localization of GFP-LC3 and LAMP1, scale bar 10 μm; (D-E) Huh7 cells co-transfected with HA-SNAP29, flag-VAMP8 and siSAC1 or siNC plasmids; (F) HepG2.2.15 cells transfected with siSAC1 or siNC 48h post transfection, co-immunoprecipitation and immunoblot analysis with the indicated antibodies; * p <0.05; * p <0.01; ns is not significant.

FIG. 8 is a schematic diagram of the molecular mechanism of the effect of SAC1 on HBV replication.

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are conventional methods unless otherwise specified; the reagents and materials used, unless otherwise indicated, are conventional in the art and are commercially available.

1 Experimental materials and reagents

1.1 Experimental materials

HepG2.2.15 cells, huh7 cells, pHBV1.3 plasmid and vector pcDNA3.1 are all commercially available. SAC1-myc plasmid: the construction of the general biological System (Anhui) is carried out by cloning the gene SACM1L (ID: 22908) -myc into the vector pcDNA3.1 (-) myc-HisA, cloning site: bamHI (GGATCC) -HindIII (AAGCTT).

1.2 Experimental reagents

1.2.1 preparation of the Main reagent

(1) 10 times electrophoretic fluid (1L)

(2) 10X transfer film liquid (1L)

(3) 5% milk sealing liquid (40 mL)

(4)10×TBS(1L)

(5)20×SSC(1L)

(6) Alkaline denaturation liquid (1L)

(7) Neutralization solution (1L)

(8) Maleic acid (1L)

(8)Washingbuffer(500mL)

(8)WashingbufferⅠ(10mL)

(9)WashingbufferⅡ(10mL)

(10)Detectionbuffer(500mL)

(11)10xTBE(1L)

(12) IP lysate

The deionized water is adopted for preparation, and the deionized water comprises the following components: 50mM Tris-HCl (pH 7.4), 50mM NaCl, 0.5% glycerol, 0.2% NP40, 1.5mM MgCl ₂ 、1×Proteinaseinhibitor。

(13) RAPA lotion

The deionized water is adopted for preparation, and the deionized water comprises the following components: 150mM KCl, 25mM Tris-HCl (pH 7.4), 5mM EDTA, 0.5% NP40, 1 XProteinaseinunder.

1.2.2 major reagent Source

1.2.3 primer or siRNA sequences

2 Experimental methods

2.1ELISA detection of supernatant and intracellular HBsAg levels

(1) Balancing the kit to room temperature, and preparing a sample to be tested in advance;

(2) Sample adding: diluting the cell supernatant according to the corresponding multiple of the cell types, adding 75 mu L of sample into each hole after uniformly mixing, and setting negative control, positive control and blank control;

(3) Incubation: sealing plates, mixing uniformly, and then placing the mixture into a constant temperature incubator at 37 ℃ for incubation for 1h;

(4) Enzyme conjugate: adding 50 mu L of enzyme conjugate into each hole, sealing the plates, uniformly mixing, and then placing the plates into a constant temperature incubator at 37 ℃ for incubation for 30min;

(5) Washing the plate: preparing a washing solution (the concentrated washing solution is diluted by ddH 2O), uncovering a sealing plate membrane, discarding the liquid, adding the washing solution after beating, standing for 30 seconds, discarding the washing solution, repeatedly washing the plate for 5 times, and beating to dry;

(6) Color development: adding 50 mu L of each of the color developing agent A and the color developing agent B into each hole, and incubating at 37 ℃;

(7) And (3) terminating: after adding 50. Mu.L of stop solution to each well and mixing, absorbance (wavelength 450 nm) was measured by an enzyme-labeled instrument, and detection data was recorded.

2.2 Western blotting experiments

2.2.1 cell lysis

(1) Collecting cell supernatant, and washing with PBS once;

(2) Adding RIPA protein lysate into 12-well plate, placing 120 μl of each well on ice, and shaking for 30min;

(3) The lysate was transferred to a 1.5mLEP tube, centrifuged at 4℃at 12000rpm for 2min;

2.2.2BCA method for detecting protein concentration

(1) Preparing a protein standard: preparing standard substances according to the specification, uniformly mixing, and performing instantaneous centrifugation, wherein the final concentration is 0.5 mug/mu L;

(2) Preparing BCA working solution: liquid volume of a: the volume of the solution B is 50:1, preparing 200 mu L of the mixture in each empty space;

(3) In a 96-well plate, 1 mu L, 2 mu L, 4 mu L, 8 mu L and 16 mu L of protein standard substances are sequentially added into a blank well, and are complemented to 20 mu L by PBS, and each sample is diluted 5 times by PBS, namely, 20 mu LPBS is added into 5 mu L of sample to be tested and is fully and uniformly mixed;

(4) Color development: after adding the standard substance and the sample to be tested, adding 200 mu LBCA working solution into each hole, and placing into a constant temperature incubator at 37 ℃ for incubation for 30min;

(5) And (3) detection: measuring absorbance (wavelength is 562 nm) by an enzyme-labeled instrument, and recording detection data;

(6) Drawing a standard curve: and drawing a standard curve by using the absorbance and the concentration of the standard substance, calculating the concentration of the sample according to the standard curve, and adjusting the concentration.

2.2.3Westernblot experiments to detect the expression level of the target protein

(1) Protein denaturation: adding 5XSDS-PAGE protein loading buffer to the protein sample with the adjusted concentration by volume, placing the protein sample in a metal bath at 95 ℃ for 10 minutes, and then placing the protein sample on ice for 10 minutes;

(2) And (3) glue preparation: cleaning a glue-making glass plate, aligning and clamping the glass plate, and preparing 10%, 12% or 15% of separation glue and 5% of concentrated glue according to the molecular size of target protein, and using after complete solidification;

(3) Loading: preventing gel into electrophoresis tank, adding 1×electrophoresis liquid to proper position, vertically pulling out comb, and adding corresponding protein sample;

(4) Electrophoresis: 80V electrophoresis for 30min, followed by 120V electrophoresis for 1h-1.5h;

(5) Transferring: after electrophoresis, cutting gel according to the molecular weight of the target protein, placing a film transfer clamp downwards according to a black surface, sequentially placing a foam cushion, thick filter paper, a gel block, a PVDF film, thick filter paper and a foam cushion, closing the film transfer clamp, placing the film transfer clamp into a film transfer instrument, and keeping constant current for 250mA film transfer for 90min;

(6) Closing: after the film transfer is finished, washing the film for five minutes by using 1 XTBST, adding 5% milk sealing liquid, and sealing for one hour at room temperature;

(7) Incubation resistance: after blocking, washing the membrane with 1 XTBE for 3 times, each for five minutes, cutting the strip according to the target protein, adding a primary antibody for incubation, and incubating overnight at 4 ℃;

(8) Washing the film: the membrane was washed 3 times with 1 XTBE for five minutes each;

(9) Secondary antibody incubation: adding a corresponding secondary antibody according to the primary antibody attribute for incubation, and incubating for 1h at normal temperature;

(10) Washing the film: the membrane was washed 3 times with 1 XTBE for five minutes each;

(11) Developing: an appropriate amount of ECL developer was added dropwise to the film, and the data were exposed and saved using a Bio-Rad gel imaging system.

2.3 quantitative qPCR (QuantitativePCR, qPCR) detection of HBVDNA levels

(1) Sample dilution: a new 200. Mu. LEP tube was prepared and 2. Mu. LHBVDNA sample and 8. Mu.L of 1 XTE buffer were added, respectively; (2) preparing a standard: a new 1.5mLEP tube was prepared and 45. Mu.L of sterilized ddH was added to each tube ₂ O, 5. Mu.L of a standard (10) ⁹ cobies/. Mu.L) was added to the first EP tube, mixed by shaking and repeated, diluted to 10 in sequence ³ copies/μL-10 ⁸ A concentration gradient of copies/. Mu.L;

(3) Preparing a PCR reaction system

(4) PCR reaction conditions

(5) And (3) calculating a PCR result: sample copy numbers were calculated according to standard curves and corresponding dilution factors.

2.4southern blot assay to detect the level of HBVDNA

(1) Sample preparation: 15 mu LHBVDNA+5 mu L of 10 XDNALoadingBuffer;

(2) Preparation of 1% agarose gel: weighing 1g agarose powder, adding 120ml of 1 XTBE buffer, putting into a microwave oven for melting, naturally cooling, pouring the gel rapidly, and standing at room temperature for about 40min until the gel is completely solidified;

(3) And (3) electrophoresis separation: adding 1 XTBE electrophoresis solution, and electrophoresis at 100V for about 90 min. Until the yellow dye runs to the edge of the glue;

(4) Alkaline denaturation and neutralization: washing with distilled water once, and denaturing for 30min; washing with distilled water once, and washing with neutral eluent (neutral buffer) for 30min; washing with distilled water once, 20 XSSC for 15min (gentle shaking);

(5) Cutting glue: a channel distance is left and right, the upper surface is cut from the middle of the blue dye and the channel, and the lower surface is cut from the lower edge of the yellow dye;

(6) Transferring: NC films of the corresponding sizes were prepared, washed with ddH2O for 5min and then soaked in 20 XSSC for 15min. The placement sequence of the film transfer device is as follows: tray (containing 20 XSSC), glass plate, thick filter paper bridging, gel, NC film (bubble is removed by glass rod), sealing film, 3 pieces of Bao Lvzhi, water absorbing paper and 2 small glass plates;

(7) Fixing: after the transfer of the membrane is finished, placing the NC membrane on filter paper for airing, and using an ultraviolet crosslinking instrument 1500J multiplied by 1 to crosslink and fix nucleic acid;

(8) Prehybridization: the prehybridization solution (DIGEasyHyb) was placed in a 42℃water bath for incubation in advance, and the pellet was dissolved. Placing the fixed NC film in a hybridization tube, adding 5ml of DIGEasyHyb buffer solution for prehybridization for 60min, and driving bubbles away by a glass rod as much as possible;

(9) Hybridization: recovering the prehybridization solution, adding denatured probe into the hybridization tube, and hybridizing at 42 ℃ overnight;

(10) Washing the film: preparing washing liquid in advance, and preheating a Washbuffer I to 42 ℃; washbuffer II is preheated to 68 ℃; washing the membrane twice with Washbuffer I for 5min each time; washing the membrane with Washbuffer II twice for 15min each time;

(11) Rinse with 20ml Washingbuffer for 5min.

(12) Closing: incubating in 15ml blockingbuffer for 30min;

(13) Incubation in 15ml anti-Digbuffer (1:10000) for 30min;

(14) Washing with 20ml Washingbuffer for 2 times each for 15min;

(15) Equilibrate in 15ml detectionbuffer for 5min;

(16) Preparing a developing solution and a fixing solution in advance, placing an NC film containing a DNA sample in a folding clamp with the NC film facing upwards, dripping a plurality of CSPD drops on the film to cover the whole film, immediately covering a sealing cover to uniformly distribute the CSPD on the film surface, preventing the generation of bubbles, and incubating for 5min at room temperature.

2.5 immunofluorescence experiments

(1) Discarding the supernatant: cells transfected for 48 hours are washed by PBS after the culture medium is sucked off, and the glass slide is picked up by an elbow needle;

(2) Washing the cells: gently dripping PBS along the edge of the slide to clean cells, tilting the cover and sucking the PBS away;

(3) Fixing formaldehyde: dripping 4% formaldehyde onto a glass slide, and fixing in a dark place for 10min;

(4) Cleaning: tilting the cover after fixing and rapidly sucking away formaldehyde, and washing with PBS for 3 times, each time for 5min;

(5) Penetrating: 1% Triton200uL was added dropwise to each slide, followed by incubation in the dark for 10min;

(6) Cleaning: tilting the cover after permeation is completed and rapidly sucking off Triton, and washing with PBS for 3 times, each time for 5min;

(7) Closing: dropwise adding 5% FBS blocking solution, and blocking for 30min;

(8) Cleaning: tilting the cover after sealing is completed, rapidly sucking away sealing liquid, and cleaning with PBS for 3 times for 5min each time;

(9) Incubation resistance: dripping the primary antibody diluted by PBS (1:200), and incubating for 1h in a dark place, so that the primary antibody can be properly prolonged;

(10) Cleaning: tilting the cover after incubation is completed and rapidly sucking away the primary antibody, and washing with PBS for 3 times, each time for 5min;

(11) Secondary antibody incubation: dripping the secondary antibody diluted by PBS (1:200), and incubating for 1h in a dark place;

(11) Cleaning: tilting the cover after incubation is completed and rapidly sucking away the secondary antibody, and washing with PBS for 3 times, each time for 5min;

(12) DAPI staining: dripping diluted DAPI reagent (1:1000) on a glass slide, and dyeing for about 10min;

(13) Cleaning: tilting the cover and rapidly sucking away DAPI after dyeing, and washing with PBS for 3 times, each for 5min;

(14) And (3) drying: drying the slide glass for about 30min;

(14) Sealing piece: dropping anti-fluorescence quencher, reversing slide, sealing with nail oil, and storing in cassette.

2.6 chronic HBV replication mouse model

2.6.1HBV Chronic replication mouse model construction

(1) Preparation of experimental animals: all animal experiments were approved by the ethical committee of Chongqing medical university (2022081). The study is strictly carried out according to the advice in the guidelines for nursing and use of laboratory animals and according to the regulations of the people's republic of China;

(2) Grouping: the purchased C57BL/6J male mice were weighed, and about 20g of the mice were selected for the experiment. Mice were first randomly divided into 2 groups: SAC1 over-expression experimental group and pcdna3.1 control group;

(3) High pressure tail vein injection: diluting the constructed pAAV-HBV1.2 plasmid and Sac1-Myc over-expression plasmid or pcDNA3.1 empty plasmid with physiological saline, injecting 2ml of plasmid diluent (containing 10 mu g of pAAV-HBV1.2 plasmid and 10 mu g of Sac1-Myc plasmid or pcDNA3.1 empty control) into each mouse by high-pressure tail vein injection, and establishing a chronic HBV replication mouse model over-expressing SAC1 genes;

(4) Collecting blood from the eyebox: mouse serum was collected on days 4, 7, 14, 21, 28, 35 and 42, respectively, after injection. Collecting liver samples of the mice at a designated time point;

(5) Sample preservation: centrifuging the serum of the mice at 8000rpm for 10min, sucking the supernatant and storing in a refrigerator at-80 ℃ for measuring the serum HBsAg level; in addition, 4% tissue cell fixative was used to preserve liver tissue samples (immunohistochemical detection of HBcAg expression in liver), and mouse liver tissue was stored in-80 refrigerator for use. The mouse carcasses are placed in a special refrigerator for animal centers for innocent treatment.

2.6.2 intrahepatic cell HBVDNA extraction

(1) Accurately weighing 60mg liver tissue, adding pre-cooled 300 μL of 1×TEbuffer (pH8.0), fully grinding, and adding 300 μL of 1×TEbuffer (pH8.0), and mixing;

(2) Add 5. Mu. LNP-40 (final concentration 0.5%), incubate on ice for 30min;

(3) Centrifuge at 14000rpm at 4℃for 1min, transfer the supernatant (approximately 900. Mu.l) to a fresh 2mLEP tube, add 5. Mu.L 1.0MMgCl2 (final concentration 5 mM) and 8. Mu.L dNaseI (10 mg/ml), incubate at 37℃for 30min;

(4) 20 μ L0.5MEDTA (final concentration 10 mM) was added;

(5) mu.L of 10% SDS (final concentration 1%) and 30. Mu.L of 20mg/mL of ropteonaseK (final concentration 0.5 mg/mL) were added and incubated at 55℃for 2 hours;

(6) Equal volumes of phenol/chloroform (500. Mu.L: 500. Mu.L, 1:1) were added and mixed by shaking. Centrifuging at 14000rpm at room temperature for 8min;

(7) The supernatant (ca. 1 mL) was transferred to a fresh 2mLEP tube, then 700. Mu.L of isopropanol (0.7V), 100. Mu.L of 3MNAAc (0.1V, pH 5.2) and 2uLtRNA (10 mg/mL) were added and placed in a-20deg.C refrigerator to precipitate overnight;

(8) After the sample was taken out of the refrigerator, it was centrifuged at 14000rpm at 4℃for 15min, and the supernatant was discarded;

(9) Adding 1mL of 75% ethanol to wash the precipitate, centrifuging at 14000rpm and 4 ℃ for 5min, and washing twice;

(10) The supernatant was discarded, the sample was transiently separated, the residual liquid was sucked off with a gun head, and then air-dried for 5 minutes, and the pellet was dissolved with 15. Mu.L of 1 XTEbuffer.

2.6.3 immunohistochemical staining (Seville organism)

(1) Paraffin sections dewaxed to water: sequentially placing the slices into dewaxing liquid I, dewaxing liquid II and dewaxing liquid III for 10min, sequentially placing absolute ethyl alcohol I (5 min), absolute ethyl alcohol II (5 min), absolute ethyl alcohol III (5 min) and washing with distilled water;

(2) Antigen retrieval: naturally cooling, placing the slide in PBS, and shaking and washing on a decolorizing shaker for 3 times, each time for 5min;

(3) Blocking endogenous peroxidases: placing the glass slide into a 3% hydrogen peroxide solution, incubating for 25min in a dark place, then placing the glass slide into PBS for decoloring treatment, and washing the glass slide on a shaking table for 3 times, wherein each time lasts for 5min;

(4) Closing: cover the slide tissue with 3% BSA, block for 30min at room temperature;

(5) Incubation resistance: draining the sealing liquid, dripping the prepared primary antibody on the slice, and placing the slice in a wet box for incubation at 4 ℃ for overnight;

(6) Secondary antibody incubation: washing the slide in PBS (phosphate buffered saline), washing on a shaker for 3 times and 5min each time, then dripping secondary antibody (HRP label) corresponding to the primary antibody, and incubating for 50min at room temperature;

(7) DAB color development: the slides were washed 3 times in PBS for 5min each. Dripping freshly prepared DAB color development liquid, controlling the color development time under a microscope, and washing the slices with tap water to terminate the color development, wherein the positive color is brown yellow;

(8) Counterstaining the nuclei: counter-dyeing with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, and washing with running water;

(9) And (3) removing the water sealing piece: sequentially placing the slices into 75% alcohol for 5min, 85% alcohol for 5min, absolute alcohol for 5min, n-butanol for 5min and xylene for 5min, dehydrating and transparency, taking out the slices from xylene, airing slightly, and sealing the slices by sealing glue.

2.7ELISA detection of intracellular PI4P levels

2.7.1 intracellular PI4P extraction

(1) Cell supernatants (12-well plates) were collected, washed once with PBS, and 150. Mu.l PBS was added to each well;

(2) Freezing in a refrigerator at-20deg.C for 1 hr, standing at room temperature for 1 hr, and vortex oscillating for one minute;

(3) Repeating the second step twice;

(4) Centrifuge at 12000rpm for 10min at room temperature, aspirate the supernatant into a new EP tube.

2.7.2ELISA detection of intracellular PI4P

(1) Dilution of standard: the original multiple standard substances in the kit are diluted into the following components in sequence: 400ng/L, 200ng/L, 100ng/L, 50ng/L, 25ng/L;

(2) Sample adding: and a blank hole, a standard hole and a sample hole to be tested are respectively arranged. Adding 50 mu L of each prepared standard substance, then adding the sample to the bottom of the ELISA plate hole, and slightly shaking and uniformly mixing without touching the hole wall as much as possible;

(3) Incubation: placing the membrane sealing plate in a constant temperature incubator at 37 ℃ for 30 minutes;

(4) Washing: preparing a washing solution (diluting the 30-time concentrated washing solution with ddH 2O), uncovering a sealing plate membrane, discarding the liquid, adding the washing solution after beating, standing for 30 seconds, discarding the washing solution, repeatedly washing the plate for 5 times, and beating to dry;

(5) Adding enzyme: adding 50 mu L of enzyme-labeled reagent into each hole;

(6) Incubation: placing the membrane sealing plate in a constant temperature incubator at 37 ℃ for 30 minutes;

(7) Washing: uncovering the sealing plate membrane, discarding the liquid, adding the washing liquid after beating, standing for 30 seconds, discarding, repeatedly washing the plate for 5 times, and beating to dry;

(8) Color development: adding 50 mu L of a color developing agent A and 50 mu L of a color developing agent B into each hole, gently shaking and uniformly mixing, and developing color at 37 ℃ for about 10 minutes in a dark place;

(9) And (3) terminating: adding 50 mu L of stop solution into each well, stopping the reaction, and measuring the absorbance (OD value) of each well at the wavelength of 450 nm; (10) calculating: and calculating a linear regression equation of the standard curve by using the concentration of the standard substance and the corresponding OD value, substituting the OD value of the sample into the equation, calculating the concentration of the sample, and multiplying the calculated concentration by the dilution multiple to obtain the actual concentration of the sample.

2.8Co-IP experiments to detect interaction between SNAP29VAMP8STX17

(1) The 10cm cell culture dish cells were further cultured for 48 hours after transfection. The cells were washed twice with PBS, digested with 1mL pancreatin (external use) for 3min, then added with 5mL complete medium (external use) to collect the cells into a 15mL centrifuge tube, and centrifuged at 4000rpm for 3min; the supernatant was aspirated off and the cell pellet was transferred to a fresh 1.5mLEP tube with 1ml DMEM complete medium (external use), centrifuged at 4000rpm for 3min, and the supernatant was aspirated off, leaving the cell pellet. (2) Adding 1ml of special cell lysate for IP on ice, and then placing in a 4 ℃ tilting table for lysis for 45min at 4 DEG C

Centrifugation at 5000rpm for 3min transferred the cell lysate supernatant to a new 1.5mLEP tube.

(3) 40ul of protein solution (Input sample) was removed to a new 1.5ml EP tube and 10ul of 5XSDS-PAGELoading Buffer was added as whole cell lysate. To the remaining samples, 2ul of the antibody of interest was added and incubated overnight at 4℃on a flip-shaker.

(4) Adding 25ul of magnetic beads into the incubated sample, washing the magnetic beads twice with 0.5% BSA in advance, re-suspending the magnetic beads with 50ul of IP lysate, adding the magnetic beads into the IP sample, and placing the mixture in a 4 ℃ turnover shaking table for incubation for 3 hours;

(5) Collecting magnetic beads with a magnetic rack, freshly configuring RAPABuffer, washing the magnetic beads with washing liquid upside down on the magnetic rack for 5 times, each time about 800ul, centrifuging at 5000rpm for 3min, and sucking the supernatant with a small gun head;

(6) With ddH ₂ O the 5xSDS-PAGELoadingBuffer is diluted into 1xSDS-PAGELoadingBuffer in advance, and added into the IP sample.

3 results

3.1SAC1 silencing promotes HBV replication

We explored whether SAC1 regulates HBV replication and viral gene expression by silencing SAC 1. To this end, hepg2.2.15 cells were transfected with two specific siRNAs against SAC1 or control siRNA, huh7 cells were co-transfected with phbv1.3 plasmid and siRNAs. 72h after transfection, we found that SAC1 silencing significantly increased the levels of hepg2.2.15 and HBV transfected Huh7 cell culture supernatant and intracellular HBsAg (fig. 1A-B). However, there was no significant effect on HBeAg and HBV RNA, including TotalHBVRNA and pgRNA. Consistent with this result, confocal microscopy showed a significant increase in the number of HBsAg punctate fluorescence after SAC1 silencing (fig. 1C). Westernblot analysis of cell lysates further showed a significant increase in the expression of hepatitis B virus core antigen (HBcAg) following SAC1 silencing (FIG. 1D). The level of HBVDNA was detected by real-time quantitative qPCR and southern blot experiments, respectively. The results showed a significant increase in intracellular HBVDNA after SAC1 silencing (fig. 1e, f). Taken together, these data indicate that SAC1 silencing promotes HBV replication and expression of related genes in HBV expressing hepatoma cells.

3.2SAC1 overexpression inhibits HBV replication

To investigate the effect of SAC1 overexpression on HBV replication and viral gene expression, we co-transfected Huh7 cells with phbv1.3 and SAC1-myc plasmid or vector pcdna3.1 expressing SAC1 gene. After 72h transfection, ELISA results showed that SAC1 overexpression significantly reduced the level of HBsAg in culture supernatants and cell lysates (FIGS. 2A-B). Confocal microscopy showed that SAC1 overexpression significantly reduced intracellular HBsAg expression in HBV transfected Huh7 cells (fig. 2C). Consistent with this result, westernblot experiments demonstrated a decrease in HBcAg expression levels after SAC1 overexpression (fig. 2D), followed by detection of HBVDNA levels using real-time quantitative qPCR and southern blot experiments. The results showed that SAC1 overexpression inhibited intracellular HBVDNA levels (fig. 2e, f). Taken together, these results indicate that SAC1 overexpression inhibits HBV replication and expression of related genes in vitro.

3.3 SAC1 overexpression inhibits HBV replication in a mouse model of chronic HBV infection

To assess the inhibition of HBV replication by SAC1 in vivo, we Hydrotropic Injected (HI) pAAV-HBV1.2 and SAC1-Myc or control pcdna3.1-Myc plasmids in a mouse model of chronic HBV infection to investigate whether SAC1 overexpression inhibited HBV replication. Mouse serum and liver samples were taken at various time points after injection (fig. 3A). SAC1 overexpression significantly inhibited HBsAg secretion for more than 6 weeks (fig. 3B). 2 or6 weeks after injection, 2 mice were sacrificed per group, livers were harvested, and intracellular HBcAg and HBVDNA expression levels were detected. Quantitative immunohistochemistry of liver sections showed that SAC1 overexpression resulted in a significant decrease in HBcAg positive hepatocytes (fig. 3C). Furthermore, real-time qPCR and southern blotting results showed that the levels of HBVDNA in mouse liver samples were significantly reduced after SAC1 overexpression (FIG. 3D-E). Taken together, these data indicate that SAC1 overexpression inhibits HBV replication in vivo.

Silencing 3.4SAC1 results in intracellular massive PI4P accumulation

Studies in yeast and mammals have shown that SAC1 is responsible for PI4P turnover in the endoplasmic reticulum and Golgi apparatus. We speculate that SAC1 may regulate HBV replication and gene expression by altering intracellular PI4P turnover. Thus, we assessed the effect of SAC1 silencing and overexpression on PI4P levels in liver cancer cell lines. Confocal microscopy showed that SAC1 silencing increased the number of PI 4P-point signals in the cell (fig. 4A), while SAC1 overexpression had the opposite effect (fig. 4B). Measurement of intracellular PI4P total levels using PI 4P-specific ELISA kits we found that SAC1 silencing increased intracellular PI4P levels (fig. 4C), whereas SAC1 overexpression reduced intracellular PI4P total levels (fig. 4D). Next, we examined the distribution of PI4P expression in golgi (gm130+), ATG9 vector (atg9+) and autophagosome (lc3+) using immunofluorescent staining. Confocal microscopy showed that SAC1 silencing significantly increased the co-localization percentage of PI4P with golgi complex (fig. 4E), ATG9 vesicle (fig. 4F), or autophagosome (fig. 4G). These results indicate that SAC1 silencing increases total PI4P levels in cells.

3.5SAC1 silencing increases HBV replication by autophagy flux

Recent studies have shown that SAC1 plays an important role in autophagy induction and autophagosome-lysosomal fusion. Our and others' studies indicate that autophagy processes affect HBV replication, whereas SAC1 plays a role in regulating autophagy, we evaluate whether SAC1 silencing would increase HBV replication by altering autophagy flux. We found that the addition of the autophagy early inhibitor VPS34-IN1 to HepG2.2.15 cells partially reversed the promotion of SAC1 silencing on supernatant and intracellular HBsAg levels (FIG. 5A). Following simultaneous treatment with autophagy inducer Torin1, the silenced SAC1, hepg2.2.15 cell supernatants and intracellular HBsAg levels were further increased (fig. 5B). These results indicate that silencing of SAC1 promotes HBV replication at least in part by altering autophagy flux. To further support our hypothesis, we assessed the effect of SAC1 silencing on the ATG5KOHuh7 cell line. Consistent with the effects of VPS34-IN1, ATG5-KO partially inhibited SAC1 silencing or overexpression from promoting or inhibiting intracellular HBcAg expression (FIGS. 5C-D), as well as supernatant and intracellular HBsAg levels (FIGS. 5E-F). Taken together, these findings indicate that SAC1 silencing increases HBV replication by modulating autophagy flux.

Silencing 3.6SAC1 results in autophagic volume accumulation

To explore how SAC1 regulates autophagy flux, we first examined the effect of SAC1 silencing or overexpression on autophagosome numbers and autophagy degradation in HBV transfected Huh7 cells. Confocal microscopy showed that SAC1 silencing significantly increased the amount of LC3 punctate fluorescence (fig. 6A), and Westernblotting analysis showed up-regulation of LC3II levels (fig. 6B). In contrast, SAC1 overexpression significantly reduced the amount of LC3 punctate fluorescence (fig. 6C), inhibiting LC3II protein expression (fig. 6D). Interestingly, SAC1 silencing significantly increased the level of cargo receptor p62, whereas SAC1 overexpression had the opposite effect. These data indicate that SAC1 silencing leads to the accumulation of large numbers of autophagosomes by interfering with autophagy degradation. Subsequently, co-transfection of Huh7 cells with mCherry-GFP-LC3 plasmid and siSAC1 or siNC with the lysosomal acidification inhibitor chloroquine as positive control resulted in accumulation of LC3 punctiform proteins expressing GFP and mCherry, similar to chloroquine treatment (fig. 6E). Taken together, these data indicate that SAC1 silencing leads to massive accumulation of autophagosomes by induction of incomplete autophagy, leading to hindered degradation of intracellular cargo.

3.7SAC1 silencing inhibits autophagosome-lysosomal fusion by blocking interactions between VAMP8 and SNAP29

Next, we studied the molecular mechanism by which SAC1 silencing leads to the accumulation of large numbers of autophagosomes in HBV expressing cells. Our recent studies indicate that SAC1 defects prevent autophagosome-lysosome fusion and, in addition, liu et al report that SAC1 deletions disrupt salmonella-containing autophagosome and lysosome fusion, thereby compromising salmonella clearance in the autophagy degradation pathway, resulting in increased bacterial replication. Based on these findings, we hypothesize that SAC1 silencing induces intracellular autophagosome accumulation by modulating autophagosome-lysosomal fusion. Thus, we studied the effect of SAC1 silencing on autophagy degradability. DQ-RedBSA degradation experiments showed that SAC1 silencing resulted in a significant decrease in the DQ-RedBSA fluorescent signal generated by intracellular autophagy lysosomal proteolysis (FIG. 7A), indicating that SAC1 silencing reduced lysosomal transport or degradation of the autophagy cargo DQ-RedBSA. To elucidate how SAC1 silencing prevents autophagy degradation, lysosomal acidification and autolysosomal formation, evaluation was performed using LysoTracker staining and immunofluorescence co-localization analysis of autophagosomes and lysosomes, respectively. Lysotracker staining showed no significant difference in red fluorescent signal of cells after SAC1 silencing (fig. 7B), indicating that SAC1 silencing did not affect lysosomal acidification. However, co-localization of GFP-LC3 and LAMP1 was significantly reduced after SAC1 silencing (fig. 7C), indicating that SAC1 silencing prevented autophagosome-lysosomal fusion. The STX17/SNAP29/VAMP8SNARE complex is critical for autophagosome fusion with lysosomes, which can recruit homologous fusion and protein sorting complexes. We studied whether SAC1 silencing inhibits autophagosome-lysosomal fusion by blocking STX17-SNAP29-VAMP8 assembly. Co-immunoprecipitation (Co-IP) experiments with either exogenous (FIG. 7D-E) or endogenous (FIG. 7F) proteins showed that SAC1 silencing blocked SNAP29 interaction with VAMP8 but not STX17 in Huh7 cells and HepG2.2.15 cells (FIG. 7D-F). Taken together, these results indicate that SAC1 silencing inhibits autophagy fusion by blocking SNAP29 binding to VAMP 8.

4 analysis

SAC1 is currently the only phosphatase that is effective in reducing PI4P levels in certain vesicles. However, whether and how SAC1 regulates HBV replication process is still unclear. We studied the effect of SAC1 on HBV replication and its underlying molecular mechanism (FIG. 8). We found that SAC1 silencing significantly increased HBV replication and HBsAg production, whereas SAC1 overexpression had the opposite effect. SAC1 silencing increased the number of autophagosomes containing HBV and PI4P levels on autophagosome membranes. In addition, SAC1 silencing prevents autophagosome-lysosomal fusion by inhibiting the interaction between SNAP29 and VAMP 8. Overall, our data indicate that phosphatase SAC1 significantly inhibits HBV replication by promoting autophagic degradation of HBV virions.

SAC1 regulates replication of certain viruses and bacteria by different mechanisms. It inhibits replication of HCV and epstein barr virus by preventing PI4P recruitment to the viral replication site. HCV has evolved strategies to destroy the antiviral effects of SAC1 in the late stages of infection. The literature reports that SAC1 deletion results in abnormal accumulation of PI4P on Salmonella-containing autophagosomes, resulting in reduced autophagy degradation and promotion of bacterial replication. Thus, SAC1 may promote bacterial and viral replication by affecting PI4P levels. Consistent with this hypothesis, our findings indicate that SAC1 silencing significantly increases HBV replication and HBsAg production in vitro, while SAC1 overexpression produces adverse effects in vitro and in a hydrodynamically injected-based mouse model of chronic HBV infection. Interestingly, studies have reported that depletion of SAC1 reduces the envelope and secretion of HBV particles in SAC1-KO Huh7 cells, while increasing the number of HBV nucleocapsids in the cells. They believe that the SAC1 deletion causes PI4P accumulation and prevents HBV envelope protein transport to the multivesicular body, suggesting SAC1 is a key host cytokine controlling HBV nucleocapsid envelope and morphogenesis. Thus, in this study we studied how SAC1 regulates other steps in HBV life cycle.

SAC 1-deficient increases the number of HBV-containing autophagosomes by up-regulating the level of its substrate PI4P on the autophagosome membrane. Following autophagy activation, SAC1 is transported from the ER to the ER-golgi intermediate region and complex where membrane components containing certain lipids are produced and then transported by ATG9 vesicles to autophagosomes for the post autophagy process. SAC1 is the only lipid phosphatase capable of hydrolyzing PI4P, reducing PI4P levels and autophagosome numbers. SAC1 and PI4P kinase phosphatidylmyo-4-kinase II alpha regulate autophagosome maturation by altering PI4P levels. Vanhauwa et al found that SAC1 affected maturation of presynaptic terminal autophagosomes. Thus, we hypothesize that SAC1 regulates HBV replication by modulating autophagy flux. We found that SAC1 silencing resulted in significantly elevated intracellular PI4P levels, mainly in golgi, ATG9 vesicles and autophagosomes. Although we cannot rule out the possibility that SAC1 defects increase PI4P levels in other organelles, our results strongly suggest that SAC1 defects modulate autophagy flux by up-regulating its substrate PI4P levels on autophagosome membranes. Further evidence is provided by the use of autophagy inhibitors VPS34-IN1 and ATG5-KOHuh7 cells (FIG. 5). The results show that autophagy inhibition partially inhibits the promotion of HBV production by SAC1 silencing, suggesting that autophagy is the primary mechanism of SAC 1-mediated HBV replication inhibition. We speculate that other effects of SAC1, including lipid transport and formation of cellular membrane scaffolds, may contribute in part to this effect in cells

Silencing of SAC1 prevents autophagosome-lysosomal fusion by inhibiting interactions between VAMP8 and SNAP29, thereby increasing HBV replication. Previous studies by Miao et al and our team showed that SAC1 silencing resulted in increased intracellular PI4P levels and prevented autophagosome-lysosomal fusion. We found that PI4P accumulation on autophagosome membranes induced by SAC1 deficiency resulted in failure of recruitment of the SNARE component of autophagosome-lysosome fusion without altering lysosomal acidification, a process that is highly conserved in yeast and eukaryotes. In agreement with this, we found that SAC1 silencing induced incomplete autophagosome formation and reduced cargo degradation by blocking autophagosome-lysosome fusion. Thus, we speculate that the main cause of SAC1 silencing leading to enhanced HBV replication and HBsAg production is impaired autophagy degradation. Since SNAP29/VAMP8/STX17SNARE complex plays a role in autophagy degradation of HBV virions, the SAC1 deletion and thus PI4P accumulated on autophagosome membrane may interfere with autophagosome-lysosomal fusion and HBV replication. Notably, accumulation of PI4P may alter the spatial conformation of the membrane. Co-IP experiments demonstrated that SAC1 silencing inhibited the interaction between SNAP29 and VAMP 8. This finding suggests that SAC1 deficiency results in PI4P accumulation in early golgi compartments, which results in excessive PI4P integration into the autophagosome membrane, defective recruitment of the SNARE component of autophagosome-lysosome fusion. Thus, SAC1 defects may increase the number of HBV autophagosomes containing excess PI4P, which are not fused to lysosomes, thereby compromising lysosomal degradation of HBV virions. However, the specific molecular mechanism by which excessive autophagosome PI4P interferes with SNARE component interactions is not yet known.

Taken together, our studies indicate that SAC1 deletion results in accumulation of large amounts of PI4P by golgi-ATG 9 vesicle-autophagosomes and blocks autophagy-lysosomal fusion by inhibiting the interaction between SNAP29 and VAMP8, thereby reducing autophagy degradation of HBV virions and enhancing HBV replication. Our results of the study provide a new strategy against HBV from the perspective of phospholipid metabolism.

Claims (5)

1. Application of SAC1 protein or recombinant plasmid expressing SAC1 protein in preparing medicine for treating hepatitis B is provided.
2. 2. The use according to claim 1, characterized in that: the recombinant plasmid for expressing the SAC1 protein is obtained by accessing a gene for encoding the SAC1 protein into an expression vector.
3. 3. The use according to claim 1, characterized in that: the recombinant plasmid is SAC1-myc plasmid.
4. 4. The use according to claim 1, characterized in that: the medicament comprises a pharmaceutically acceptable carrier.
5. 5. The use according to claim 1, characterized in that: the medicine is tablet, capsule, injection, granule, powder, pill or oral liquid.