Direct Targeting of the Nucleocapsid Protein by Morin Inhibits Hantaan Virus Infection

Direct Targeting of the Nucleocapsid Protein by Morin Inhibits Hantaan Virus Infection | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 20 September 2025 V1 Latest version Share on Direct Targeting of the Nucleocapsid Protein by Morin Inhibits Hantaan Virus Infection Authors : Yi Xing 0009-0007-4895-7636 , Yuexi Zhao , Lulu Luo , Yaoxuan Qiu , Wei Ye 0000-0002-3980-8547 , Liang Zhang , Xuemin Pei , Hongwei Ma 0000-0003-4929-3222 , Fanglin Zhang , and Linfeng Cheng [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175835175.54764377/v1 Published Biochemical and Biophysical Research Communications Version of record Peer review timeline 242 views 301 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Hantaan virus (HTNV), a major etiologic agent of hemorrhagic fever with renal syndrome (HFRS), poses a significant threat to global public health. Currently, there are no clinically approved antiviral drugs specifically for HTNV infection, nor are there any highly effective preventive vaccines. Thus, the development of effective therapeutic strategies is critically important. This study investigated the antiviral efficacy of morin against HTNV and its potential mechanism of action using in vitro assays. Our results demonstrate that morin inhibits HTNV infection in a concentration-dependent manner and primarily targets the post-entry replication stage of the viral life cycle. Further mechanistic studies revealed that morin exerts its antiviral effects by directly interacting with the HTNV nucleocapsid protein (NP). In conclusion, this study identifies morin as a promising candidate for anti-HTNV drug development, providing a new strategic direction for HFRS treatment. Direct Targeting of the Nucleocapsid Protein by Morin Inhibits Hantaan Virus Infection Yi Xing 1,2* , Yuexi Zhao 2* , Lulu Luo 2* ，Yaoxuan Qiu 2 , Wei Ye 2 , Liang Zhang 2 , Xuemin Pei 2 ，Hongwei Ma 2 , Fanglin Zhang 2 , Linfeng Cheng 2 （1.School of Medical Technology，Shaanxi University of Chinese Medicine，Xianyang，Shaanxi 712046，China；2.School of Basic Medicine，Air Force Medical University，Xi’an，Shaanxi 710032，China） Correspondence: Hongwei Ma ( [email protected] ), Fanglin Zhang ( [email protected] ), Linfeng Cheng ( [email protected] ) Funding: The present study was supported by grants from the National Natural Science Foundation of China (No. 82371835). Keywords: HTNV，HFRS，Morin，Flavonoid，Antiviral ABSTRACT: Hantaan virus (HTNV), a major etiologic agent of hemorrhagic fever with renal syndrome (HFRS), poses a significant threat to global public health. Currently, there are no clinically approved antiviral drugs specifically for HTNV infection, nor are there any highly effective preventive vaccines. Thus, the development of effective therapeutic strategies is critically important. This study investigated the antiviral efficacy of morin against HTNV and its potential mechanism of action using in vitro assays. Our results demonstrate that morin inhibits HTNV infection in a concentration-dependent manner and primarily targets the post-entry replication stage of the viral life cycle. Further mechanistic studies revealed that morin exerts its antiviral effects by directly interacting with the HTNV nucleocapsid protein (NP). In conclusion, this study identifies morin as a promising candidate for anti-HTNV drug development, providing a new strategic direction for HFRS treatment. 1 | Introduction Hantaan virus (HTNV) is a negative-sense RNA virus belonging to the genus Orthohantavirus within the Hantaviridae family [1] . Its genome consists of three segments—large, medium, and small—which encode the RNA-dependent RNA polymerase (RdRp), the glycoprotein precursor (GPC), and the nucleocapsid protein (NP), respectively. The GPC is subsequently cleaved proteolytically into two envelope glycoproteins, Gn and Gc [2] . HTNV is the etiologic agent of hemorrhagic fever with renal syndrome (HFRS), a disease that causes tens of thousands of infections globally each year, particularly in East Asia and Europe, and has a case fatality rate ranging from 5% to 15% [3, 4] . HFRS is characterized by acute fever, renal impairment, hemorrhagic manifestations, and hypotensive shock. Severe cases may progress to multiple organ failure [5] . Current clinical management relies primarily on supportive care, including fluid resuscitation, renal replacement therapy (e.g., hemodialysis), and management of symptoms. To date, no specific antiviral drugs have been approved for the treatment of HTNV infection [6-8] . Therefore, the development of effective and specific antiviral agents against HTNV remains an urgent public health priority. Morin is a natural flavonoid compound widely present in plants of the Moraceae family, as well as in various traditional Chinese medicines including mulberry fruit, mulberry leaf, and perilla [9, 10] It has been demonstrated to exhibit diverse biological activities, including antioxidant [11] , anti-inflammatory [12, 13] anticancer [14] and hepatorenal protective effects [15, 16] Previous studies have reported its inhibitory effects against influenza viruses [17, 18] , however, its activity against HTNV has not been explored. Given this context, investigating its potential anti-HTNV properties is of considerable interest. This study aimed to investigate the antiviral efficacy of morin against HTNV and to elucidate its underlying mechanism of action. Our findings demonstrate that morin effectively inhibits HTNV replication in a concentration-dependent manner, primarily during the post-entry stage of the viral life cycle. Further mechanistic studies indicate that morin likely exerts its antiviral effect by directly interacting with the HTNV nucleocapsid protein (NP). In summary, these findings identify morin as a promising candidate for the development of therapeutics against HTNV infection. 2 | Materials and Methods 2.1 | Cells and virus The human lung adenocarcinoma cell line A549 and the African green monkey kidney cell line Vero-E6 were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; BBI, Shanghai, China) supplemented with 10% or 2% fetal bovine serum (FBS; Procell, Wuhan, China, Cat# 164210), along with 1% penicillin-streptomycin (P/S; BBI, Cat# E607009-0500) solution, and incubated at 37°C in a humidified 5% CO₂ atmosphere. Cells were washed with Dulbecco’s phosphate-buffered saline (DPBS; BBI, Cat# E607009). HTNV (Strain 76-118) was propagated and titrated in Vero-E6 cells in our laboratory and stored at –80°C until use. 2.2 | Compounds Morin (purity ≥98%) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China, Cat# A135920). The compound was stored desiccated and protected from light at -20°C. For use, it was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA, Cat# D5879) to generate a 200 mM stock solution. The stock solution was aliquoted and stored at –80°C. 2.3 | Virus preparation and infection To amplify the viral stock, Vero-E6 cells were infected with HTNV. Infection was performed when the cells reached approximately 70% confluence. After 5-7 days, the culture supernatant was harvested, clarified by centrifugation (4,000 × g, 10 min, 4°C) to remove cellular debris, aliquoted, and stored at -80°C for future use. For infection experiments, cells were seeded into culture plates. When the cells reached 60-70% confluence, they were washed twice with DPBS before infection. The cells were infected with HTNV at a multiplicity of infection (MOI) of 0.1. After adsorption at 37°C for 2 h, the viral inoculum was removed, and the cells were gently washed once with DPBS and then overlaid with fresh DMEM supplemented with 2% FBS. The infected cells were then cultured for 24 h before being processed for subsequent experiments according to the specific requirements of each assay. 2.4 | Cytotoxicity Assay The cytotoxicity of morin on A549 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay. A549 cells were seeded into 96-well plates and divided into three groups: the blank control (medium only), the negative control (cells with medium), and the drug-treated group (cells with various concentrations of morin). The blank control wells contained 100 µL of DMEM supplemented with 10% FBS without cells. The negative control wells contained cells in 100 µL of DMEM with 10% FBS. The drug-treated wells contained cells treated with 100 µL of morin at serial concentrations (12.5, 25, 50, 100, 200, 400, and 800 µM) prepared in DMEM with 10% FBS. After incubation for 48 h at 37°C in a 5% CO₂ atmosphere, 10 µL of CCK-8 reagent (Targetmol, Cat# C0005) was added to each well, followed by incubation in the dark at room temperature for 1 h. The absorbance (optical density, OD) at 450 nm was then measured using a BioTek Epoch microplate reader. Cell viability was calculated using the following formula: Viability (%) = [(ODₛ - OD₆) / (ODₙ - OD₆)] × 100, where ODₛ is the absorbance of the drug-treated well, ODₙ is the absorbance of the negative control well, and OD₆ is the absorbance of the blank control well. 2.5 | Analysis of Viral Infection Inhibition A549 cells were seeded in 12-well plates and infected with HTNV (MOI = 0.1) as described in the ’Virus preparation and infection’ section. During viral adsorption, the cells were treated with various concentrations of morin (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μM) simultaneously. After the 2 h adsorption period, the inoculum was removed, and the cells were overlaid with fresh maintenance medium (DMEM with 2% FBS) containing the corresponding concentration of morin. After 24 h of incubation, the cells were harvested, and total RNA was extracted. The intracellular RNA levels of the HTNV S segment were quantified by quantitative real-time PCR (qRT-PCR). The expression level of the HTNV S segment in the untreated control group (0 μM morin) was defined as 100%. The half-maximal inhibitory concentration (IC₅₀) of morin against HTNV-S gene expression was calculated using GraphPad Prism software 10.1.2. 2.6 | Time Course Analysis of Morin’s Activity Against HTNV A time-of-addition assay was performed to delineate the stage-specific antiviral effect of morin (200 μM). The experiment comprised six treatment groups, differentiated by the timing of drug administration relative to the time of infection (defined as t = 0 h):Group 1 (Full-course): Morin was present from 1 h pre-infection (t = -1 h) to 24 hpi (t = 24 h).Group 2 (Pre-treatment): Morin was present from t = -1 h to t = 0 h and then removed. Group 3 (Co-treatment): Morin was present only during the viral adsorption period (t = 0 h to t = 2 h). Groups 4-6 (Post-treatment): Morin was added at 2 h, 6 h, or 12 hpi and maintained until t = 24 h. The virus control group (HTNV infection only) was processed identically but without any morin treatment. After the 2 h adsorption period, the inoculum was replaced with maintenance medium (DMEM with 2% FBS), and cells were cultured until 24 hpi. for sample collection. 2.7 | Dose-Response Assay A549 cells were treated with increasing concentrations of morin (50, 100, and 200 μM). Treatment was initiated simultaneously with HTNV infection (MOI = 0.1). The cells were then incubated for 24 h at 37°C in a 5% CO₂ atmosphere. 2.8 | Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from harvested cells using the Super FastPure Cell RNA Isolation Kit (Vazyme, Nanjing, China, Cat# RC101) according to the manufacturer’s protocol. Subsequently, 1 μg of total RNA was reverse-transcribed into cDNA using the 4× All-in-One Ultra qRT SuperMix (Vazyme, Cat# R05). The 20 μL reverse transcription reaction system consisted of 1 μg RNA, 10 μL of 4×All-in-One Ultra qRT SuperMix, and RNase-free ddH₂O to a final volume of 20 μL. The reactions were incubated under the following conditions: 50°C for 10 min (reverse transcription), followed by 85°C for 5 sec (enzyme inactivation). qRT-PCR was performed using 2× qPCR SmArt Mix (Yeasen, Shanghai, China, Cat# 10101) on a Bio-Rad C1000 real-time PCR system. The reactions were used to quantify the mRNA levels of the HTNV S segment and the endogenous control gene 18S rRNA. The primer sequences used were as follows:HTNV S segment: Forward: 5’-GAGCCTGGAGACCATCTG-3’,Reverse: 5’-CGGGACGACAAAGGATGT-3’.18S rRNA: Forward: 5’-GTAACCCGTTGAACCCCATT-3’,Reverse: 5’-CCATCCAATCGGTAGTAGCG-3’. 2.9 | Western blotting Cells were harvested at the indicated time points and lysed on ice for 30 min using RIPA lysis buffer (Beyotime, Shanghai, China, Cat# P0013C) supplemented with a protease inhibitor cocktail (Targetmol, Shanghai, China, Cat# C0001). The protein concentration of the lysates was quantified using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Cat# 23227). Equal amounts of protein (20 μg per lane) were denatured by boiling at 95°C for 5 min in 1× Laemmli sample buffer. The denatured proteins were separated electrophoretically on a 10% SDS-polyacrylamide gel (NCM Biotech, Suzhou, China, Cat# P2011) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Cat# ISEQ00010) via the wet transfer method. The membrane was blocked with 5% non-fat milk in TBST for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer: anti-HTNV monoclonal antibody (1A8, prepared by the Department of Microbiology and Pathogen Biology, Air Force Medical University，1:1000 dilution) and anti-β-actin (Mouse mAb, Immunoway, Plano, TX, USA, Cat# PTR2364, 1:5000 dilution). After washing, the membrane was incubated with appropriate IRDye-conjugated secondary antibodies (Li-Cor Biosciences, 1:10,000 dilution) for 1 h at room temperature protected from light. Protein bands were visualized and quantified using an Odyssey CLx infrared imaging system (Li-Cor 5200). Band intensities were quantified using Image software and normalized to β-actin. 2.10 | Immunofluorescence Assay (IFA) Following infection and treatment, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. After washing with PBS, cells were blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The cells were then incubated overnight at 4°C with the primary anti-HTNV monoclonal antibody (1A8) diluted in PBS. The next day, after washing, cells were incubated for 1 h at room temperature in the dark with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Sangon Biotech, Shanghai, China, Cat# D110090) diluted in 3% BSA. Following another wash, cell nuclei were counterstained with Hoechst 33258 (MedChemExpress, Monmouth Junction, NJ, USA, Cat# HY-15558, 1 μg/mL) for 10 min at room temperature. Images were captured using an IX71 inverted fluorescence microscope (Olympus) under consistent exposure settings for all comparative analyses. 2.11 | Focus formation assay(FFA) Vero E6 cells seeded in 96-well plates were inoculated in parallel with 100 µL of undiluted and serially diluted (10-fold steps) cell culture supernatants from each experimental group. Each dilution was tested in quadruplicate. After a 2 h adsorption period at 37°C, the inoculum was removed and the cells were overlaid with a semisolid maintenance medium consisting of DMEM, 2% FBS, and 1.6% carboxymethyl cellulose (CMC).Following a 6-day incubation at 37°C, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS for 10 min, and then blocked with 3% BSA in DPBS for 1 h at room temperature to prevent nonspecific binding. The plates were then incubated overnight at 4°C with the primary anti-HTNV antibody (1A8) diluted in DPBS. Subsequently, the plates were incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) secondary antibody (ABclonal, Woburn, MA, USA, Cat# AS003) diluted in DPBS. Finally,TMB substrate(Biokits, Cat# WB003) was added to each well for color development in the dark. The foci in each well were counted manually or by using an immunospot reader [19] .The viral titer was calculated in focus-forming units per milliliter (FFU/mL) using the following formula: FFU/mL = (N × D) / V, where N is the average number of foci per well, D is the dilution factor, and V is the volume of inoculum per well (0.1 mL in this case) 2.12 | Molecular docking The three-dimensional structures of the following HTNV-related proteins: NP (PDB ID: 5FSJ), RdRp (PDB ID: 8C4S), Gn (PDB ID: 6Y6P), and Gc (PDB ID: 5LJY), were retrieved from the Protein Data Bank in PDB format. Subsequently, the crystal structures were processed using PyMOL to eliminate crystalline water molecules and heterologous ligands. Following this, polar hydrogen atoms were added, and Gasteiger charges were assigned using AutoDock Tools 1.5.7, resulting in the final PDBQT format files for docking. The structure of the ligand, morin (PubChem CID: 5281670), was obtained from the PubChem database in SDF format and subsequently converted to PDB format using Open Babel 2.4.1. The ligand structure was then imported into AutoDock Tools to define rotatable bonds before generating its PDBQT file. Molecular docking simulations were carried out using AutoDock Vina 1.1.2 under a semi-flexible model, wherein the protein backbones were treated as rigid bodies, while the side chains within the binding site and all rotatable bonds in the ligand were permitted to rotate freely. The conformation exhibiting the lowest binding energy and belonging to the most populous cluster was selected as the optimal binding mode for further analysis. Finally, the resulting docking poses were visually inspected and analyzed using PyMOL and LigPlot+ to elucidate molecular interactions and to generate schematic diagrams of the binding modes. 2.13 | Surface Plasmon Resonance(SPR) The HTNV NP (MedChemExpress, HY-P76965) was covalently immobilized on a CM5 sensor chip (Cytiva) via amine coupling to create a stable surface. A 100 mM stock solution of morin was prepared in DMSO and subsequently subjected to serial dilution in the running buffer to achieve a concentration series ranging from 6.25 to 100 μM. All measurements were performed at 25 °C using HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v surfactant P20, pH 7.4) supplemented with 1% (v/v) DMSO to mitigate bulk refractive index artifacts and minimize nonspecific binding. Analyte injections were performed in duplicate at a flow rate of 30 μL/min. The association phase was monitored for 120 s, followed by a 300 s dissociation phase in running buffer. 2.14 | MicroScale Thermophoresis(MST) HTNV NP (MedChemExpress, HY-P76965) was fluorescently labeled using the His-Tag Labeling Kit RED-tris-NTA 2nd Generation. A stock solution of morin was subjected to serial dilution in assay buffer to create a concentration gradient. The labeled protein was incubated with varying concentrations of morin, and the resulting ligand-protein complexes were loaded into standard treated capillaries. The fluorescence changes induced by a temperature gradient were then recorded on a Monolith NT.115 instrument (NanoTemper Technologies). The dissociation constant (KD) of the morin–HTNV NP complex was determined from the analysis of the dose-response curves derived from the concentration-dependent changes in thermophoretic mobility. 2.15 | Pull-down assay Morin was custom-biotinylated by Bioty Science Co., Ltd. The resulting biotin-morin conjugate was aliquoted and stored protected from light at -20 °C to preserve stability. A549 cells were cultured in T75 flasks and infected with HTNV at ~70% confluence. Following a 24 h incubation, the cells were harvested and lysed, followed by centrifugation at 4 °C to collect the soluble supernatant (cell lysate). The cell lysates were divided into two aliquots: one was incubated with biotin-morin (200 μM final concentration), whereas the control aliquot was incubated with an equimolar amount of free biotin. Each mixture was then incubated with streptavidin-coated magnetic beads overnight at 4 °C under constant rotation. After incubation, the beads were immobilized using a magnetic rack, washed extensively with DPBS, and bound complexes were eluted using 1× Laemmli loading buffer. Finally, the eluted samples were denatured by boiling for 5 min and analyzed by Western blotting for the detection of HTNV NP. 2.16 | RNA sequencing (RNA-seq) A549 cells were infected with HTNV and subsequently incubated with morin for 24 h. Two experimental groups were established: an HTNV-infected group treated with morin and an HTNV-infected, drug-untreated control group. After the 24-hour incubation, cells from each group were lysed with 1 mL of TRIzol™ reagent (Tiangen Biotech, DP424) per well of a 6-well plate. The lysates were immediately snap-frozen in liquid nitrogen and stored at −80 °C until further processing. Total RNA was extracted from all samples, followed by library construction and high-throughput sequencing. These services were performed by Wuhan Servicebio Technology Co., Ltd. 2.17 | Statistical analysis All data were analyzed statistically and visualized using GraphPad Prism 10.1.2. All experiments were independently repeated at least three times, and data are expressed as the mean ± standard deviation (SD). Dose-response curves were generated by fitting the data to a four-parameter logistic (4PL) nonlinear regression model to determine the IC₅₀. Statistical comparisons between groups were performed using one-way analysis of variance (ANOVA), with significance defined as * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. A P value greater than 0.05 was considered not statistically significant (NS). 3 | Results 3.1 | Effect of Morin on A549 cell viability and anti-HTNV activity The chemical structure of morin is shown in Fig. 1A. The cytotoxicity of morin in A549 cells was evaluated using the CCK-8 assay. Treatment with morin at concentrations as high as 400 µM did not significantly reduce cell viability, suggesting negligible cytotoxicity at these concentrations (Fig. 1B). Therefore, a concentration of 200 µM was chosen as the maximum safe dose for subsequent experimental studies. The inhibitory effect of morin on HTNV infection was comprehensively assessed across a range of eight concentrations (from 3.125 to 400 µM). Morin inhibited HTNV replication in a dose-dependent manner (Fig. 1C). The half-maximal inhibitory concentration (IC₅₀) value was calculated to be 8.03 µM by fitting the data to a four-parameter logistic (4PL) model. Figure.1 Morin inhibits HTNV infection in A549 cells without cytotoxicity. (A) The chemical structure of morin. (B) Cytotoxicity assessment. Viability of A549 cells treated with morin at the indicated concentrations for 48 h was measured using the CCK-8 assay. Data are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for comparison with the untreated control group; ns, not significant; * P < 0.05. (C) Antiviral activity of morin against HTNV. HTNV-infected A549 cells (MOI = 0.1) were treated with the indicated concentrations of morin. At 24 hpi, viral RNA levels in the culture supernatant were quantified by qRT-PCR. Data were normalized to 18S rRNA and are presented as relative expression to the infected, untreated control (set to 100%) using the 2^(–ΔΔCt) method. The IC₅₀ is indicated. 3.2 | Inhibitory effect of morin on HTNV infection at different time points in A549 cells. To identify the stages of the HTNV life cycle targeted by morin, a time-of-addition assay was conducted in A549 cells. The antiviral efficacy was assessed by quantifying both HTNV NP expression and viral progeny production (titer). Six treatment regimens were established, differing in the timing of 200 μM morin administration relative to HTNV infection, as schematically detailed in Fig. 2A. As shown in Fig. 2B-D, both viral titer and NP expression were most profoundly suppressed in Group 1 (full-course treatment) and Group 6 (treatment initiated at 12 hpi), demonstrating that morin exerts its most potent antiviral effect when present throughout the viral life cycle or when administration begins at the 12 h time point. A significant reduction in viral titer was also observed in Groups 3, 4, and 5 (Fig. 2B), alongside more variable decreases in HTNV NP expression (Fig. 2C, D). Collectively, these results indicate that morin inhibits HTNV at multiple stages of its replication cycle, with a particularly strong effect on post-entry events. The most potent inhibition was achieved with either full-course treatment or with administration commencing at 12 hpi. Figure.2 The inhibitory effect of morin on HTNV infection at different time points in A549 cells. (A) Schematic of the experimental timeline for morin treatment relative to HTNV infection. (B) Viral titers in the culture supernatants, as determined by FFA. Cells were infected with HTNV (MOI = 0.1) and treated with morin at the indicated times. Supernatants were harvested 24 hpi, and viral titers were measured to assess the inhibitory effect of morin across different treatment schedules. Data are presented as the mean ± SD; n = 4. Statistical significance was analyzed by one-way ANOVA; ****, P < 0.0001; ns, not significant. (C) Analysis of HTNV NP expression by Western blotting. Cells were infected and treated as described in (B). Total protein was extracted 24 hpi for Western blotting to evaluate the effect of morin on HTNV NP expression. Quantification of Western blotting results (mean ± SD; n = 3). Statistical analysis was performed using one-way ANOVA; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (D) Immunofluorescence analysis of A549 cells infected and treated as in (B), stained for HTNV NP (green) and nuclei (blue). Representative images were acquired using an IX71 fluorescence microscope. Scale bar=50 μm. 3.3 | Morin inhibits HTNV replication in a dose-dependent manner. A549 cells were treated with morin (50, 100, or 200 µM) for the duration of the HTNV infection. The control group consisted of HTNV-infected cells without morin treatment (0 µM). Analysis of viral titers showed a gradual decrease with increasing morin concentrations compared to the control (Fig. 3A). Similarly, morin treatment at all concentrations (50, 100, and 200 µM) significantly reduced HTNV NP expression (Fig. 3C, D). Furthermore, the mRNA level of the HTNV S segment was significantly decreased after treatment with 100 or 200 µM morin (Fig. 3B). These results indicate that morin inhibits HTNV infection in a dose-dependent manner, with higher concentrations exerting more potent antiviral effects. Figure.3 Dose-dependent inhibitory effect of morin on HTNV infection. (A)Viral titers in the cell supernatants were measured by a FFA.A549 cells were infected with HTNV (MOI = 0.1) and simultaneously treated with morin at the indicated concentrations for 24 h. The supernatants were collected, and viral titers were quantified as focus-forming units per milliliter (FFU/mL); data are shown as the mean ± SD (n = 4). Statistical analysis was performed by one-way ANOVA; *** P < 0.001, **** P < 0.0001. (B) The mRNA expression level of the HTNV S segment was analyzed by qRT-PCR. Cells were infected and treated as described above, followed by total RNA extraction and quantification of the viral RNA; results are expressed as the mean ± SD (n = 3). Statistical significance was assessed by one-way ANOVA; ns: not significant, * P < 0.05, ** P < 0.01. (C)HTNV NP expression was analyzed by Western blotting. Cells were infected and treated as described above. At 24 hpi, total protein was extracted and subjected to Western blotting; quantitative results are presented as the mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA; *** P < 0.001, **** P < 0.0001. (D) Representative immunofluorescence images of HTNV NP (green) and nuclei (blue) in A549 cells. Cells were infected and treated as described above. Images were acquired using an IX71 fluorescence microscope. Scale bar = 50 μm. 3.4 | Predicted binding of morin to HTNV proteins via molecular docking We used molecular docking to predict the interactions between morin and various HTNV proteins. The molecular docking results showed that the binding energies of morin to HTNV NP, RdRp, Gn, and Gc were -8.6, -8.3, -8.1, and -7.7 kcal/mol, respectively. Morin formed two hydrogen bonds with residues ASP307 and TRP310 of HTNV NP (Fig. 4A), four with THR1163, SER970, THR1174, and TYR713 of HTNV RdRp (Fig. 4B), three with GLU26, THR122, and THR342 of HTNV Gn (Fig. 4C), and two with TRP47 and GLY44 of HTNV Gc (Fig. 4D). Among these targets, morin exhibited the strongest binding affinity (lowest binding energy) for HTNV NP, suggesting that the morin-NP complex has the highest stability. Although morin formed more hydrogen bonds with RdRp, Gn, and Gc, their binding energies were less favorable than that with NP. A comprehensive analysis of the binding energies and hydrogen-bonding patterns revealed that NP provided the most favorable free energy landscape for morin binding. The key hydrogen bonds were located in the core region of NP, which is a critical area for nucleocapsid assembly. These results suggest that morin may preferentially target HTNV NP, potentially exerting antiviral effects by stabilizing the NP conformation and interfering with viral nucleocapsid formation. Figure.4 Molecular docking of morin with HTNV proteins. (A) Morin in complex with HTNV NP. (B) Morin in complex with HTNV RdRp. (C) Morin in complex with HTNV Gn. (D) Morin in complex with HTNV Gc. All complex structures were visualized using PyMOL (left and upper right panels) and analyzed using LigPlot+ to generate two-dimensional interaction diagrams (lower right panels). In the 3D views, HTNV proteins are depicted as a translucent blue molecular surface, morin is shown as a green stick model, and hydrogen bonds are indicated by yellow dashed lines with relevant residues labeled. The 2D interaction diagrams summarize hydrogen bonds (green dashed lines) and hydrophobic interactions (red arcs). 3.5 | Morin binds HTNV NP in vitro and in cellulo SPR analysis showed that morin binds to HTNV NP in a specific and dose-dependent manner, with a KD of 47.6 μM (Fig. 5A). The interaction was further validated by MST. In this assay, the mobility of HTNV NP decreased progressively with increasing morin concentrations. An S-shaped binding curve was fitted, yielding a KDvalue of 43.9 μM, which is consistent with a specific binding interaction between morin and HTNV NP (Fig. 5B). Additionally, in vitro pull-down assays demonstrated a significant enrichment of HTNV NP by biotin-morin, but not by biotin alone, providing direct evidence for a specific protein-ligand interaction between morin and HTNV NP (Fig. 5C). Figure.5 Experimental validation of the direct interaction between morin and HTNV NP. (A) SPR analysis. HTNV NP was immobilized on a CM5 sensor chip, and a series of concentrations of morin were injected as the analyte for kinetic analysis. The resulting sensorgram displayed typical concentration-dependent binding curves. The KD was determined by fitting the data to a 1:1 binding model. (B) MST analysis. Fluorescently labeled HTNV NP was used as the target, and a series of concentrations of morin were titrated as the ligand. The fluorescence-time trajectories showed concentration-dependent changes in the thermophoretic signal. An S-shaped binding curve was generated from the fitted data, providing a KD value. (C) Pull-down assay. The left panel illustrates the structure of biotin-morin. The right panel shows that biotin-morin or free biotin (as a control) was incubated with streptavidin-coated magnetic beads and HTNV NP for 36 h. After washing, the captured complexes were analyzed by Western blotting. 3.6 | Screening of HTNV targets and pathways inhibited by morin This study confirms that morin exerts an antiviral effect by directly targeting the HTNV NP. However, whether it also relies on host factors to exert its full antiviral effect remains unclear. To investigate this, we employed high-throughput RNA-seq to comprehensively analyze the transcriptomic profiles of HTNV-infected cells with or without morin treatment, focusing on morin-regulated signaling networks and immune responses. Differential gene expression analysis using the DESeq2 method identified 6907 differentially expressed genes (DEGs), comprising 3289 up-regulated and 3618 down-regulated genes (Fig. 6A). Functional annotation revealed that these DEGs were predominantly enriched in specific biological processes (Fig. 6B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were significantly enriched in the Toll-like receptor, tumor necrosis factor (TNF), and interleukin-17 (IL-17) signaling pathways, as well as in cytokine-cytokine receptor interactions and viral infection-related pathways such as Hepatitis B and Measles (Fig. 6C). These pathways are primarily involved in viral recognition, innate immune activation, inflammatory responses, and cytokine signaling. This suggests that morin may mitigate HTNV infection and pathogenesis by modulating these host antiviral and inflammatory signaling axes. To identify key host genes potentially regulated by morin, we performed cluster analysis on the top 200 DEGs (Fig. 6D). This analysis revealed that four host genes—NEAT1, IL7R, CLEC5A, and EPSTI1—exhibited the most pronounced expression changes in response to morin treatment. Among these, NEAT1, IL7R, and CLEC5A were up-regulated, while EPSTI1 was down-regulated. This suggests that morin may modulate HTNV infection by regulating the expression of these host factors. Figure.6 Transcriptomic analysis of host gene expression profiles regulated by morin during HTNV infection. (A) Volcano plot of DEGs. The plot displays the distribution of DEGs in A549 cells infected with HTNV and treated with morin. The x-axis represents the log₂(fold change), and the y-axis represents the -log₁₀(p-value). Red dots denote significantly up-regulated genes, and blue dots represent significantly down-regulated genes. (B) Gene Ontology (GO) enrichment analysis. The DEGs are categorized into the three main GO domains: biological process, cellular component, and molecular function. (C) KEGG pathway enrichment analysis. DEGs were mapped to the KEGG database. The top 15 significantly enriched pathways ( p < 0.05) are shown. The dot size corresponds to the number of DEGs in each pathway, and the color intensity (from blue to red) indicates the level of statistical significance, with red representing a higher significance level. The x-axis shows the enrichment ratio (number of DEGs in pathway / total number of genes in pathway), and the y-axis lists the pathway names. (D) Heatmap of the top 200 DEGs. The heatmap displays the expression patterns of the top 200 DEGs in A549 cells infected with HTNV and treated with morin. The x-axis represents the different experimental samples, and the y-axis represents the clustered genes. The color gradient from blue to white to pink indicates low to high expression levels (z-score), with pink and blue representing high and low expression, respectively. 4 | Discussions Hantaan virus (HTNV) is the primary etiological agent of hemorrhagic fever with renal syndrome (HFRS) [20] . Given the continuous expansion of endemic areas, the annual rise in case numbers, and the current lack of approved specific antiviral drugs, HFRS poses a significant threat to global public health [21, 22] . Therefore, elucidating the pathogenic mechanisms of HTNV and developing safe, effective targeted antiviral therapies are imperative to reduce HFRS mortality and improve patient prognosis. In this study, we systematically evaluated the flavonoid morin for its inhibitory activity against HTNV infection. Our results demonstrated that morin significantly suppressed HTNV replication in A549 cells, with a half-maximal inhibitory concentration (IC₅₀) of 8.03 μM (Fig. 1). To identify the stage of the viral life cycle targeted by morin, the compound was administered at various time points relative to infection. Treatment with morin at 2, 6,or 12 hours post-adsorption consistently inhibited HTNV replication. The most pronounced inhibitory effect was observed when morin was present throughout the entire infection cycle—from pre-adsorption to the replication phase (Fig. 2). These findings indicate that morin primarily acts on post-entry stages of the HTNV life cycle. Furthermore, the anti-HTNV activity of morin was concentration-dependent, as shown by a gradual reduction in viral replication levels with increasing concentrations of the compound (Fig. 3). During HTNV infection, virions bind to cell surface receptors and enter host cells via endocytosis. Subsequently, the ribonucleoprotein complex (RNP) is released into the cytoplasm to initiate viral genome transcription and replication [23] As a core component of the viral replication machinery, the HTNV nucleocapsid protein (NP) specifically binds viral RNA to form helical nucleocapsids and provides a replication template for the viral RNA-dependent RNA polymerase (RdRp) [24] .Furthermore, NP facilitates the release of virions from host cells by modulating the translation of host valine-containing proteins, thereby promoting viral spread to neighboring cells [25] . These multifunctional roles of NP in viral assembly and host immune regulation establish it as a promising target for antiviral drug development. To elucidate the molecular mechanism underlying the anti-HTNV activity of morin, we performed molecular docking simulations between morin and all HTNV structural and non-structural proteins. The results indicated that morin exhibits binding potential to all viral proteins, with the strongest binding affinity observed for the NP, as reflected by the lowest binding energy (Fig. 4). This interaction was further validated using surface plasmon resonance (SPR), microscale thermophoresis (MST), and pull-down assays. Both SPR and MST consistently demonstrated that morin binds to HTNV NP in a dose-dependent manner, with dissociation constants (KD) of 47.6 μM and 43.9 μM, respectively. Additionally, pull-down assays confirmed that biotin-morin specifically enriched HTNV NP, whereas no detectable signal was observed in the blank control group (Fig. 5). In summary, this study systematically demonstrates the anti-HTNV activity of morin at the cellular level and establishes that its antiviral effect is mediated through a direct interaction with HTNV NP. Although it is established that morin directly targets HTNV NP, whether its antiviral activity involves host factors remains unclear. To address this question, we employed RNA sequencing (RNA-seq) to analyze transcriptomic changes in HTNV-infected cells following morin treatment, with a particular focus on altered signaling networks and immune responses. The RNA-seq results revealed significant alterations in host gene expression after morin treatment, suggesting a potential association with the regulation of antiviral innate immunity and inflammatory processes. Notably, the upregulation of NEAT1 [26] , IL7R [27] , and CLEC5A [28] may play important roles in mediating these immunoregulatory effects. Currently, research on the anti-HTNV effects of morin is limited to the in vitro level, as existing HTNV infection models fail to stably recapitulate the typical pathological processes of HFRS. This limitation is primarily attributable to the absence of robust animal models that faithfully recapitulate the typical pathological processes of HFRS [29] . Conventional immunocompetent mouse models of HTNV infection generally remain asymptomatic [30, 31] , whereas immunodeficient nude mice can develop fatal disease but exhibit substantial differences from human immune responses [32] ，Although non-human primate models offer higher clinical relevance, their use is constrained by high costs, specialized facility requirements, and ethical considerations, rendering such studies currently infeasible. Therefore, future efforts should prioritize developing more physiologically relevant animal model systems to enable rigorous in vivo evaluation of morin’s efficacy and mechanisms of action. In summary, this study identifies morin as a promising anti-HTNV drug candidate. Its unique mechanism of action supports its further development as a therapeutic agent. Future research should focus on in vivo assessment and further mechanistic investigation to fully exploit morin’s therapeutic potential against HTNV. Author Contribution Yi Xing: writing-original draft, investigation, formal analysis, data curation, visualization, validation. Yuexi Zhao: methodology, investigation. Lulu Luo: methodology, investigation. Yaoxuan Qiu: formal analysis, investigation . Wei Ye: methodology, conceptualization, supervision. Liang Zhang: project administration, resources. Xuemin Pei: formal analysis, investigation, validation . Hongwei Ma: methodology, conceptualization, supervision. Fanglin Zhang: conceptualization, supervision, funding acquisition. Linfeng Cheng : supervision, writing-reviewing and editing, conceptualization, funding acquisition. Acknowledgments The present study was supported by grants from the National Natural Science Foundation of China (No. 82371835). The funding bodies had no role in the design, interpretation, or submission of this work for publication. Consent This work does not involve the use of human subjects and animals. 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Keywords antiviral agents epidemiology hantavirus virus classification zoonoses Authors Affiliations Yi Xing 0009-0007-4895-7636 Shaanxi University of Chinese Medicine View all articles by this author Yuexi Zhao Air Force Medical University View all articles by this author Lulu Luo Air Force Medical University View all articles by this author Yaoxuan Qiu Air Force Medical University View all articles by this author Wei Ye 0000-0002-3980-8547 Air Force Medical University View all articles by this author Liang Zhang Air Force Medical University View all articles by this author Xuemin Pei Air Force Medical University View all articles by this author Hongwei Ma 0000-0003-4929-3222 Air Force Medical University View all articles by this author Fanglin Zhang Air Force Medical University View all articles by this author Linfeng Cheng [email protected] Air Force Medical University View all articles by this author Metrics & Citations Metrics Article Usage 242 views 301 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yi Xing, Yuexi Zhao, Lulu Luo, et al. 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