Application of NCOA4 gene / NCOA4 protein expression inhibitors in the preparation of drugs to inhibit rotavirus replication

Abstract

This invention discloses the application of an NCOA4 gene / NCOA4 protein expression inhibitor in the preparation of drugs to inhibit rotavirus replication, relating to the fields of biomedicine and antiviral therapy. This invention uses an NCOA4 gene / NCOA4 protein expression inhibitor to prepare drugs to inhibit rotavirus replication, effectively suppressing rotavirus replication by inhibiting NCOA4 gene / NCOA4 protein expression.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and antiviral therapy, specifically to the application of an NCOA4 gene / NCOA4 protein expression inhibitor in the preparation of drugs that inhibit rotavirus replication. Background Technology

[0002] Rotavirus (RV) is a major pathogen causing severe viral diarrhea in infants and young animals, resulting in a significant disease burden and economic losses worldwide. Currently, clinical interventions primarily rely on fluid support therapy and vaccination, lacking specific antiviral drugs that directly target viral replication. Therefore, elucidating the novel pathogenic mechanisms of rotavirus and developing new therapeutic targets is of significant clinical importance and application value.

[0003] Ferroptosis is an iron-dependent form of regulated cell death characterized primarily by the accumulation of lipid peroxides. Recent studies have found that various viruses can induce ferroptosis to promote their own replication by disrupting the iron homeostasis of host cells. Ferritin autophagy is one of the core regulatory mechanisms in this process. In this pathway, nuclear receptor coactivator 4 (NCOA4) acts as a key selective autophagy receptor, specifically mediating the transport and degradation of ferritin (especially its heavy chain subunit FTH1) into autolysosomes, thereby releasing stored iron ions. Intracellular free Fe 2+ When levels rise, reactive oxygen species (ROS) can be generated through the Fenton reaction, which can trigger fatal lipid peroxidation and ultimately lead to ferroptosis.

[0004] Although the role of ferroptosis in viral infection is gradually being recognized, whether rotavirus infection regulates host cell ferroptosis through the NCOA4-mediated ferritin autophagy pathway, thereby affecting viral replication efficiency, remains unreported, and the specific molecular mechanism is also unknown. This knowledge gap limits the possibility of developing new anti-rotavirus strategies targeting this pathway. Summary of the Invention

[0005] The purpose of this invention is to provide an application of an NCOA4 gene / NCOA4 protein expression inhibitor in the preparation of a drug to inhibit rotavirus replication, which effectively inhibits rotavirus replication by inhibiting the expression of the NCOA4 gene / NCOA4 protein.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides the application of NCOA4 gene / NCOA4 protein expression inhibitors in the preparation of drugs to inhibit rotavirus replication.

[0007] Furthermore, the rotavirus is a porcine intestinal rotavirus.

[0008] Furthermore, the NCOA4 gene / NCOA4 protein expression inhibitor is at least one of the following: siRNA targeting the NCOA4 gene, shRNA targeting the siRNA of the NCOA4 gene, antisense oligonucleotides targeting the siRNA of the NCOA4 gene, antibodies targeting the NCOA4 protein, antigen-binding fragments targeting the NCOA4 protein, and small molecule compounds that inhibit NCOA4 transcription / translation.

[0009] Furthermore, the sense and antisense strand sequences of the siRNA targeting the NCOA4 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, or the sense and antisense strand sequences of the siRNA targeting the NCOA4 gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0010] In a second aspect, the present invention provides a pharmaceutical composition for inhibiting rotavirus replication, comprising the above-mentioned NCOA4 gene / NCOA4 protein expression inhibitor and a pharmaceutically acceptable excipient, wherein the NCOA4 gene / NCOA4 protein expression inhibitor is at least one of the following: siRNA targeting the NCOA4 gene, shRNA targeting the siRNA of the NCOA4 gene, antisense oligonucleotide targeting the siRNA of the NCOA4 gene, antibody targeting the NCOA4 protein, antigen-binding fragment targeting the NCOA4 protein, and small molecule compound that inhibits NCOA4 transcription / translation.

[0011] Thirdly, the present invention provides the application of autophagy inhibitors that inhibit NCOA4 in the preparation of drugs that inhibit rotavirus replication.

[0012] Furthermore, autophagy inhibitors are used to suppress the NCOA4-mediated ferritin autophagy pathway to inhibit rotavirus replication.

[0013] Furthermore, the autophagy inhibitors are 3-methyladenine and / or chloroquine.

[0014] Fourthly, the present invention provides a pharmaceutical composition for inhibiting rotavirus replication, comprising the above-mentioned 3-methyladenine or chloroquine.

[0015] The present invention has the following beneficial effects: This invention uses IPEC-J2 cells infected with rotavirus as a model. By knocking down or overexpressing NCOA4 and treating with autophagy inhibitors, it was demonstrated that rotavirus infection leads to intracellular free Fe by upregulating NCOA4 expression, promoting the co-localization of NCOA4 and LC3, and mediating the autophagic degradation of ferritin FTH1 and FTL. 2+Increased concentration and ROS accumulation induce ferroptosis and enhance viral replication. Furthermore, this invention also found that knocking down NCOA4 expression or blocking autophagy flux using autophagy inhibitors 3-methyladenine or chloroquine can effectively inhibit ferritin degradation and Fe2+ degradation induced by RV infection. 2+ The release and ROS generation significantly improved the survival rate of infected cells and significantly inhibited the expression of rotavirus protein VP6 and viral replication. This invention provides a novel drug target and strategy for anti-rotavirus therapy, and has significant clinical application value and development prospects. Attached Figure Description

[0016] Figure 1 To detect the expression level of NCOA4 mRNA in IPEC-J2 cells after transfection with different NCOA4 siRNAs by RT-qPCR; Figure 2 To detect the expression level of NCOA4 protein in IPEC-J2 cells after transfection with different NCOA4 siRNAs using Western Blot; Figure 3 The results of quantitative analysis of NCOA4 protein in IPEC-J2 cells after transfection with different NCOA4 siRNAs; Figure 4 To detect the cell viability of IPEC-J2 cells transfected with NCOA4 siRNA using the CCK-8 assay; Figure 5 To detect the effect of NCOA4 knockdown on the viability of IPEC-J2 cells infected with RV using the CCK-8 assay; Figure 6 To detect the effect of NCOA4 knockdown on RV VP6 protein expression using immunofluorescence; Figure 7 The results of quantitative analysis of RV VP6 protein after NCOA4 knockdown; Figure 8 To detect the expression level of RV VP6 protein after NCOA4 knockdown using Western blotting; Figure 9 To detect the expression level of RV VP6 mRNA after NCOA4 knockdown by RT-qPCR; Figure 10 FerroOrange fluorescent probe to detect the effect of NCOA4 knockdown on intracellular Fe in RV-infected cells 2+ The influence of level; Figure 11 To detect the effect of NCOA4 knockdown on intracellular ROS levels after RV infection using the DCFH-DA fluorescent probe; Figure 12To detect the effect of NCOA4 knockdown on the colocalization level of NCOA4 and LC3 after RV infection using immunofluorescence assay; Figure 13 To detect bands in Western Blot of the knockdown of NCOA4 on the expression levels of ferroptosis-related proteins FTH1, FTL, and LC3-II after RV infection; Figure 14 Quantitative analysis of FTH1, FTL, and LC3-II protein expression levels; Figure 15 To detect the expression level of NCOA4 mRNA in IPEC-J2 cells after NCOA4 overexpression by RT-qPCR; Figure 16 Western blot was used to detect the expression level of NCOA4 protein in IPEC-J2 cells after NCOA4 overexpression. Figure 17 The results of quantitative analysis of NCOA4 protein in IPEC-J2 cells after overexpression of NCOA4; Figure 18 The effect of NCOA4 overexpression on the viability of IPEC-J2 cells was detected using the CK-8 assay. Figure 19 The effect of NCOA4 overexpression on the viability of IPEC-J2 cells infected with RV was detected by CCK-8 assay. Figure 20 To detect the effect of NCOA4 overexpression on RV VP6 protein expression using immunofluorescence; Figure 21 Western blot was used to detect the expression level of RV VP6 protein after NCOA4 overexpression; Figure 22 The results of quantitative analysis of RV VP6 protein after overexpression of NCOA4; Figure 23 To detect the expression level of RVVP6 mRNA after NCOA4 overexpression by RT-qPCR; Figure 24 FerroOrange fluorescent probe was used to detect the effect of NCOA4 overexpression on intracellular Fe in RV-infected cells. 2+ The influence of level; Figure 25 The DCFH-DA fluorescent probe was used to detect the effect of NCOA4 overexpression on intracellular ROS levels after RV infection; Figure 26 To detect the effect of NCOA4 overexpression on the co-localization level of NCOA4 and LC3 after RV infection using immunofluorescence; Figure 27Western blot was used to detect bands showing the expression levels of ferroptosis-related proteins FTH1, FTL, and LC3-II after RV infection by overexpression of NCOOA4. Figure 28 Quantitative analysis of the expression levels of FTH1, FTL, and LC3-II proteins. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions made based on the spirit of the present invention without departing from its content and scope are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0018] The IPEC-J2 cells (porcine small intestinal epithelial cell line) used in this embodiment were purchased from the National Experimental Cell Resource Sharing Platform. Cells were cultured in DMEM / F12 medium (Gibco) containing 10% fetal bovine serum (Excell) and 1% penicillin-streptomycin (Beyotime), and incubated at 37 ℃ with 5% CO2 saturated humidity. Rotavirus (RV) OSU (G5P7) strain (ATCC#VR-893) was purchased from the National Veterinary Microbiological Culture Collection Center. Before use, the virus was activated with trypsin (25 μg / mL) at 37 ℃ for 30 min. When the IPEC-J2 cells reached 80% confluence, they were infected with RV virus solution with a multiplicity of infection (MOI) of 10. After 1 hour of adsorption, the medium was replaced with maintenance medium (serum-free DMEM / F12 containing 1% penicillin-streptomycin) and cultured for another 24 hours for subsequent experiments.

[0019] In a specific embodiment of this invention, the siRNA targeting the NCOA4 gene was synthesized by Gemma Biotechnology Co., Ltd. Three siRNAs (siNCOA4-1, siNCOA4-2, and siNCOA4-3) and a negative control (siCtrl) were designed; the specific sequences are shown in Table 1. Before transfection, IPEC-J2 cells were seeded in culture plates. When the cell confluence reached 70%-80%, transfection was performed according to the Lipofectamine 3000 (Invitrogen) instructions. Lipofectamine 3000 and Opti-MEM (Gibco) were mixed at a ratio of 1:20 and incubated at room temperature for 5 min. Simultaneously, the siRNA stock solution (20 μM) was mixed with Opti-MEM at a ratio of 1:20 and equilibrated at room temperature for 5 min. The two solutions were then mixed in equal volumes and incubated at room temperature for 15 min to form a nucleic acid-liposome complex. The cell culture medium was removed, the cells were washed with PBS, and the complex was added. Opti-MEM was then added to the final volume to achieve a final siRNA concentration of 20 nM. Twelve hours after transfection, the medium was replaced with complete culture medium, and the cells were cultured further.

[0020] Table 1. siRNA sequences targeting the NCOA4 gene In a specific embodiment of this invention, the NCOA4 overexpression plasmid pcDNA3.1-NCOA4 was synthesized by Beijing Qingke Biotechnology Co., Ltd. IPEC-J2 cells were seeded in culture plates, and when the cell confluence reached 70%-80%, the plasmid was transfected into the cells according to the Lipofectamine 3000 instructions. Twenty-four hours after transfection, the cells were infected with RV at 10 MOI for 24 hours for subsequent detection.

[0021] In a specific embodiment of this invention, cell viability was detected using an enhanced CCK-8 assay kit (Beyotime). IPEC-J2 cells were seeded in 96-well plates (1×10⁻⁶ cells / wells). 4 Cells / well), after appropriate treatment, were added to each well with 110 μL of CCK-8 working solution (basal medium and CCK-8 mixed at a 10:1 ratio) and incubated at 37 ℃ for 2 hours. Absorbance at 450 nm and 650 nm was measured using a full-wavelength microplate reader, with 650 nm as the reference wavelength. Relative cell viability was calculated using the following formula: Viability (%) = [(Experimental group OD value - Blank group OD value) / (Control group average OD value - Blank group OD value)] × 100%.

[0022] In a specific embodiment of the present invention, the immunofluorescence staining step is as follows: IPEC-J2 cells are seeded in a 24-well plate (5×10⁻⁶ cells / well). 4Cells / wells were treated accordingly, then the culture medium was removed, washed with pre-cooled PBS, and fixed with 4% paraformaldehyde at 4 °C for 15 min. After washing three times with PBS, the cells were permeated with 0.5% Triton X-100 (Beyotime) at room temperature for 10 min. After washing three times with PBS, the cells were blocked with blocking buffer containing 1% goat serum (Beyotime) and 0.3% Triton X-100 at room temperature for 60 min. The blocking buffer was discarded, and primary antibodies (anti-VP6 antibody, Abcam, 1:200; anti-NCOA4 antibody, Santa Cruz, 1:200; anti-LC3 antibody, Zenbio, 1:200) were added and incubated overnight at 4 °C. The next day, after washing with PBS, the cells were incubated with the corresponding fluorescent secondary antibody (DyLight 488 or CY3 labeled, Boster, 1:1000) and Hoechst 33342 nuclear dye (Seville, 1:1000) at room temperature in the dark for 60 min. After washing with PBS, the slides were mounted with anti-fluorescence quenching mounting medium and observed and images were acquired using an inverted fluorescence microscope (Olympus IX53).

[0023] In a specific embodiment of this invention, intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe (ThermoFisher). IPEC-J2 cells were seeded in 24-well plates, treated accordingly, and then the culture medium was removed. Cells were washed with pre-warmed PBS, and a balanced salt solution containing 20 μM DCFH-DA was added. The cells were incubated at 37 °C in the dark for 30 min. After washing three times with PBS, Hoechst 33342 nuclear staining solution (1 μg / mL) was added, and the cells were incubated in the dark for 20 min. Immediately after washing with PBS, the cells were observed and images were acquired using a fluorescence microscope.

[0024] In a specific embodiment of the present invention, intracellular Fe 2+ Horizontal detection was performed using the FerroOrange fluorescent probe (Dojindo). IPEC-J2 cells were seeded in 24-well plates, treated accordingly, washed with PBS, and stained with 1 μM FerroOrange working solution at 37 °C in the dark for 20 min. After washing with PBS, the cells were observed and images were acquired using a fluorescence microscope (excitation 543 nm / emission 580 nm).

[0025] In a specific embodiment of this invention, the total RNA extraction and real-time quantitative PCR (RT-qPCR) procedures are as follows: Total RNA was extracted from cells using RNAiso Plus (TaKaRa) according to the manufacturer's instructions. RNA purity and concentration were detected using NanoDrop 2000. cDNA was synthesized using HiScript III RT SuperMix (Novizan) under the following conditions: incubation at 42 °C for 15 min, followed by inactivation at 85 °C for 5 s. qPCR was performed using TB Green Premix Ex Taq II (TaKaRa) on a Bio-Rad real-time PCR instrument. The reaction program was: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing for 30 s (annealing temperatures are shown in Table 2), extension at 72 °C for 10 s, for a total of 42 cycles; followed by melting curve analysis. β-actin was used as an internal reference gene, and 2... - The relative expression level of the target gene was calculated using the ΔΔCt method. The primer sequences and annealing temperatures used are shown in Table 2.

[0026] Table 2 Primer sequences used in this embodiment In a specific embodiment of this invention, the total protein extraction and Western blotting procedures are as follows: Total cellular protein was extracted using RIPA lysis buffer (Strong, Beyotime). 1% protease / phosphatase inhibitor mixture (Beyotime) and 1 mM PMSF (Beyotime) were added to the lysis buffer. After lysis on ice for 10 min, the mixture was centrifuged at 4 ℃ and 12000×g for 10 min, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit (Beyotime). The protein sample was mixed with 5×SDS-PAGE loading buffer at a ratio of 4:1 and denatured in a boiling water bath for 5 min. 20 μg of protein was loaded, separated by SDS-PAGE gel electrophoresis, and then wet-transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 2 h, and then incubated overnight at 4 ℃ with primary antibody (dilution shown in Table 3). The next day, the membrane was washed 5 times with TBST, and then incubated at room temperature for 1.5 h with HRP-labeled secondary antibody (Cell Signaling Technology or Zenbio, 1:5000). After washing, the membrane was developed using ECL chemiluminescence solution (Beyotime), and images were acquired using a gel imaging system (Bio-Rad). The gray values ​​of the bands were analyzed using ImageJ software, and the relative expression level of the protein was calculated using β-actin as an internal reference.

[0027] Table 3 Antibody information from Western Blot experiments In a specific embodiment of the present invention, data statistics and analysis were performed using the following methods: experimental data are expressed as mean ± standard error (Mean ± SEM). SPSS 20.0 software was used for statistical analysis. Two-tailed t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Duncan's method was used for post-hoc multiple comparisons. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered extremely statistically significant.

[0028] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0029] Example 1: Effects of knockdown of NCOA4 expression on rotavirus replication and ferroptosis (1) Verification of NCOA4 siRNA knockdown efficiency: In this embodiment, three siRNAs targeting NCOA4 (siNCOA4-1, siNCOA4-2, and siNCOA4-3) were designed. RT-qPCR results showed that, compared with the negative control (siCtrl), all three siRNAs significantly reduced the expression level of NCOA4 mRNA, with siNCOA4-3 showing the highest knockdown efficiency. Figure 1 Western blot analysis further confirmed that siNCOA4-3 significantly downregulated NCOA4 protein expression levels. Figure 2 , Figure 3 CCK-8 cell viability assays showed that transfection with siNCOA4-3 had no significant effect on the viability of IPEC-J2 cells. Figure 4 ).

[0030] (2) Effect of NCOA4 knockdown on RV replication: To detect whether NCOA4 knockdown affects RV replication, the expression levels of RV VP6 protein and mRNA were detected by immunofluorescence, Western blotting, and RT-qPCR. Immunofluorescence results showed that the fluorescence intensity of VP6 protein was strong in the RV-infected group, while the fluorescence intensity of VP6 was significantly reduced after NCOA4 knockdown ( Figure 6 Western blot and RT-qPCR results further showed that knocking down NCOA4 significantly inhibited VP6 protein expression levels. Figure 7 , Figure 8 ) and mRNA expression level ( Figure 9 Furthermore, CCK-8 assays showed that knocking down NCOA4 significantly alleviated the decrease in cell viability caused by RV infection. Figure 5 The above results indicate that knocking down NCOOA4 expression can effectively inhibit rotavirus replication.

[0031] (3) Effects of NCOA4 knockdown on ferroptosis-related indicators: To further investigate whether NCOA4 knockdown affects RV replication by inhibiting ferroptosis, intracellular Fe was measured. 2+ Intracellular Fe levels, ROS levels, co-localization of NCOA4 and LC3, and expression of ferritin-related autophagy proteins were measured. The FerroOrange fluorescent probe was used to detect intracellular Fe levels. 2+ Levels, results showed that RV infection led to Fe 2 + The fluorescence intensity was significantly enhanced, and knockdown of NCOA4 effectively suppressed this phenomenon. Figure 10 (Scale bar 50 μm). Similar results were obtained from detecting ROS levels using the DCFH-DA fluorescent probe. Figure 11 Immunofluorescence colocalization assays showed that RV infection promoted the colocalization of NCOA4 and the autophagy marker LC3, and the colocalization signal was weakened after NCOA4 knockdown. Figure 12 Western blot analysis of ferritin autophagy-related protein expression showed that RV infection led to FTH1 and FTL protein degradation and increased LC3-II expression, while knockdown of NCOA4 significantly reversed these changes. Figure 13 , Figure 14 The above results indicate that knocking down NCOA4 reduces Fe by inhibiting ferritin autophagy. 2+ It releases and accumulates ROS, thereby inhibiting RV replication and alleviating cell damage.

[0032] Example 2: Effects of NCOOA4 overexpression on rotavirus replication and ferroptosis (1) Verification of NCOA4 overexpression efficiency: To further verify the key role of NCOA4, an NCOA4 overexpression plasmid (pcDNA3.1-NCOA4) was constructed in this embodiment. RT-qPCR results showed that the intracellular NCOA4 mRNA level was significantly increased after NCOA4 overexpression ( Figure 15 Western blot analysis further confirmed a significant upregulation of NCOA4 protein expression levels. Figure 16 , Figure 17 CCK-8 assay showed that overexpression of NCOOA4 had no significant effect on the viability of IPEC-J2 cells. Figure 18 ).

[0033] (2) Effect of NCOA4 overexpression on RV replication: Immunofluorescence detection results showed that the fluorescence intensity of VP6 protein was significantly enhanced after NCOA4 overexpression compared with the RV infection group. Figure 20 Western blot and RT-qPCR results further showed that overexpression of NCOA4 significantly promoted VP6 protein expression levels. Figure 21 , Figure 22 ) and mRNA expression level ( Figure 23 CCK-8 assay showed that overexpression of NCOA4 further exacerbated the decrease in cell viability induced by RV infection. Figure 19 These results, in turn, confirm the important role of NCOA4 in promoting RV replication.

[0034] (3) Effects of NCOA4 overexpression on ferroptosis-related indicators: FerroOrange fluorescent probe detection results showed that NCOA4 overexpression could further enhance intracellular Fe induced by RV infection. 2+ accumulation( Figure 24 The DCFH-DA probe yielded similar results in detecting ROS levels. Figure 25 Immunofluorescence colocalization assays showed that overexpression of NCOA4 promoted the colocalization of NCOA4 and LC3 after RV infection. Figure 26 Western blot analysis showed that overexpression of NCOOA4 further exacerbated FTH1 and FTL degradation and LC3-II upregulation induced by RV infection. Figure 27 , Figure 28 In summary, overexpression of NCOA4 promotes rotavirus replication by enhancing ferritin autophagy-dependent ferroptosis.

[0035] In summary, rotavirus infection promotes its own replication by upregulating NCOA4 expression and activating the NCOA4-mediated ferritin autophagy-ferroptosis axis. Knocking down NCOA4 expression effectively blocks this pathway and significantly inhibits rotavirus replication. Therefore, NCOA4 and its mediated ferritin autophagy pathway are ideal targets for anti-rotavirus therapy.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of NCOA4 gene / NCOA4 protein expression inhibitors in the preparation of drugs to inhibit rotavirus replication.

2. Use according to claim 1, characterized in that, The NCOA4 gene / NCOA4 protein expression inhibitor is at least one of the following: siRNA targeting the NCOA4 gene, shRNA targeting the NCOA4 gene siRNA, antisense oligonucleotides targeting the NCOA4 gene siRNA, antibodies targeting the NCOA4 protein, antigen-binding fragments targeting the NCOA4 protein, and small molecule compounds that inhibit NCOA4 transcription / translation.

3. Use according to claim 2, characterized in that, The sense and antisense sequences of the siRNA targeting the NCOOA4 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, or the sense and antisense sequences of the siRNA targeting the NCOOA4 gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. A pharmaceutical composition for inhibiting replication of rotavirus, characterized by, The invention includes the NCOA4 gene / NCOA4 protein expression inhibitor of claim 2 and a pharmaceutically acceptable excipient, wherein the NCOA4 gene / NCOA4 protein expression inhibitor is at least one of the following: siRNA targeting the NCOA4 gene, shRNA targeting the siRNA of the NCOA4 gene, antisense oligonucleotides targeting the siRNA of the NCOA4 gene, antibodies targeting the NCOA4 protein, antigen-binding fragments targeting the NCOA4 protein, and small molecule compounds that inhibit NCOA4 transcription / translation.

5. Application of autophagy inhibitors that inhibit NCOA4 in the preparation of drugs to inhibit rotavirus replication.

6. The application according to claim 5, characterized in that, The autophagy inhibitor is used to inhibit the NCOA4-mediated ferritin autophagy pathway to suppress rotavirus replication.

7. The application according to claim 5, characterized in that, The autophagy inhibitor is 3-methyladenine and / or chloroquine.

8. A pharmaceutical composition for inhibiting rotavirus replication, characterized in that, Includes 3-methyladenine or chloroquine as described in claim 7.

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