IntroductionViruses, particularly RNA viruses, cause many serious outbreaks and pandemics around the world, resulting in high mortality rates and economic problems1. According to the World Health Organization (WHO), 58 and 296 million people are infected with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infections, respectively. Viral hepatitis (VH) is responsible for almost one million fatalities worldwide, with HBV and HCV accounting for 820,000 and 290,000 deaths, respectively2,3. Despite the availability of an active and effective HBV vaccine, all available therapies to treat HBV are not able to completely remove the virus or eradicate the integrated covalently closed circular DNA (cccDNA), but rather inhibit viral replication4,5. The new treatment for HCV using direct acting antivirals (DAAs) is very effective and targets three proteins: non-structural (NS) NS3/NS4A protease, NS5A region, and NS5B polymerase6. However, the high cost of these drugs remains a key barrier to their widespread use7.The most challenging part of selecting antiviral medicines is determining an effective and safe antiviral drug that does not destroy host cells8. Furthermore, the advent of untreatable illnesses and microorganism resistance to available antimicrobial medications enhance the demand for novel drugs. Antiviral screening of natural compounds and their derivatives is a promising source9,10. These sources are rich in bioactive chemicals with unique chemical structures that might be used to develop new antiviral drugs11. Mushrooms are a rich source of proteins, glucans, vitamins, ergosterol and important minerals with anti-tumor, antiproliferative, and immunomodulatory activity12,13. Lectins are non-immunoglobulin glycoproteins that are capable of binding to a wide range of sugar molecules with high selectivity and stereo specificity without changing the ligands of glycosyl14. Lectins have a role in cellular signaling, cancer, glycoprotein scavenging from the circulatory system, cell–cell interaction in the immune cells, differentiation, and protein binding to cellular components, as well as host defense, inflammation, and metastatic mechanisms15. Mushroom lectins have gotten a lot of attention recently because of their anti-proliferative, antibacterial, and immunomodulatory characteristics16,17,18. The aim of this study was to purify and characterize a lectin from Pleurotus ostreatus (POL), evaluate its in vitro antiviral activity against HBV and HCV, and investigate its potential mechanisms of action. The result can emphasize the usefulness of the mushrooms and its lectin content as a natural source of antiviral compounds and may open a window to discover alternative agents to substitute the current antiviral products and finding novel compounds that can fight the emerged viral diseases.ResultsMushroom lectin (POL) purificationThe elution profiles obtained from the mono-Q column using FPLC for the crude extract are depicted in Fig. 1a. The fractions displaying a notable protein concentration (14, 15, and 16) were collected. To assess their lectin content, a hemagglutination (HA) test was performed, and subsequently, these fractions were loaded onto the fetuin affinity column. Figure 1b; Table 1 showcase the results of the fetuin column elution and purification steps of lectin, respectively. The elution performed with 0.5 M NaCl demonstrated the highest concentration at 280 nm, measuring 0.835, along with an HA titer of 4096 units of lectins. This was followed by an elution utilizing 0.75 M NaCl. The molecular weight of the purified lectin was determined through SDS-PAGE (Fig. 2A), illustrating the progression of the purification steps for P. ostreatus. In this regard, lane “2” distinctly displayed the purified POL, characterized by a singular band with an approximate molecular weight of 45 kDa. Additionally, an analysis using native gel electrophoresis with the purified POL (depicted in Fig. 2B) revealed the presence of a solitary band, indicating a molecular weight of approximately 90 kDa. This comparison underscores the variations in molecular weight based on the separation technique employed.Fig. 1(a) A typical FPLC elution profile for the chromatography of POL on mono-Q column previously equilibrated with phosphate buffer (pH = 7.4), eluted with the same buffer containing 1 M NaCl. The elution was at flow rate 3 mL/fraction and absorbance measured at 280 nm. (b) Fetuin affinity purification of POL.Full size imageTable 1 Purification scheme of POL shows that using ammonium sulphate at final concentration 60%, resulting in POL specific activity of 97.52 u/mg, and 41.63 purification fold increases in purity.Full size tableFig. 2Protein gel electrophoresis of P. ostreatus mushroom using (A) 15% SDS PAGE: lane 1; protein ladder, lane 2; eluted protein from fetuin column, lane 3; eluted protein from mono Q column, lane 4; 60% (NH4)2SO4 precipitate, lane 5; crude extract, and (B) Native gel electrophoresis: lane 6; eluted protein from mono Q column, lane 7; crude extract, lanes 8 and 9; eluted protein from fetuin column.Full size imageCharacterization of the purified lectin (POL) from mushroomTable 2 illustrates the hemagglutination (HA) activity of the purified POL. It is noteworthy that the highest titers were observed, measuring 4096 HU/mL for the human group O and 256 HU/mL for the AB group.Table 2 Hemagglutination units of the purified POL.Full size tableThe hemagglutination inhibitory activity (HAI) of POL was unaffected by monosaccharides or disaccharides. However, melibiose and fetuin were able to inhibit POL activity, with minimum inhibitory concentrations of 100 and 6.25 mM, respectively. POL displayed its highest hemagglutination activity at pH 6 and 7, both registering at 100%. Conversely, its activity was negligible at pH 1, 2, 3, 10, 11, and 12, each recording 0% compared to the control. These findings indicate that POL activity remains stable up to 40 °C, decreasing to 50% at 60 °C, and ceasing entirely at 80 °C compared to the control. Furthermore, the impact of various metal cations on lectin activity was assessed. In Table S1, it is shown that the hemagglutination titer of the purified lectin was unaffected by the addition of Mg2+, K2+, Ca2+, Ba2+, Zn2+, Mn2+, and Al3+ ions. However, the activity of POL was dose-dependently inhibited upon the addition of Fe3+ (6–10 mM).Evaluation of the purified lectin cytotoxicityThe dose-response relationship for the cytotoxic effect of POL on both the Vero cell line and PBMCs is illustrated in Table S2. Additionally, the cytotoxicity of POL was evaluated on other cell lines, including Huh-7 and HepG2. It is noteworthy that the impact of POL on both normal and cancer cells followed a dose-dependent pattern. The data obtained from the experiments indicated that incubating POL with the Vero and PBMC cell lines showed minimal cytotoxic activity, as indicated in Table 3 by the 100% effective concentration (EC100) values of 3.14 and 2.96 µM, respectively. Furthermore, Table 3 presents the IC50 values of POL against the human hepatoma cancer cells HepG2 and Huh-7 used in this study, which were 68.20 and 38.21 µM, respectively. These findings emphasize the lack of significant cytotoxicity of POL towards normal cell lines under the tested conditions.Table 3 The IC50 and EC100 values of POL against PBMCs, Vero, HepG2 and Huh-7 cell lines.Full size tableEvaluation of antiviral activity of POLThe antiviral activity of the purified lectin against HCV and HBVIn Fig. 3a and Supplementary Table S3, it is demonstrated that POL exhibits promising anti-HCV effects, particularly when employed in blocking and neutralizing mechanisms. The inhibition percentages of the lectin showed dose-dependency. In the context of positive control infected Huh-7 cells, the HCV titer increased from 2.9 × 104 to 3.3 × 106 copies/mL. Notably, the application of 12.5 µM POL resulted in the complete prevention of HCV entry into Huh-7 cells. Impressively, POL displayed the most notable efficacy in the blocking and neutralizing mechanisms, as evidenced by its significantly lower IC50 concentrations of 68.75 and 52.125 nM, respectively, compared to the standard anti-HCV drug sofosbuvir (SOF). Unlike POL, sofosbuvir exhibited a high IC50 value of 357.28 nM in the treatment mechanism, without demonstrating any preventive effects in the blocking or neutralization mechanisms. Additionally, it is important to note that POL exhibited a notably high selectivity index (SI) value of 2360.69 in its neutralizing effect, indicating a substantial margin between protein cytotoxicity and antiviral efficacy. In Fig. 3b and Supplementary Table S4, POL demonstrated promising anti-HBV effects, displaying increased potency when engaging treatment and blocking mechanisms. Similarly, the percentages of lectin inhibition displayed a dependence on dosage. In the scenario involving positive control infected HepG2 cells, the HBV titer increased from 1.0 × 104 to 6.69 × 105 copies/ml. Intriguingly, the utilization of 12.5 µM POL exhibited the ability to prevent both HBV entry and replication within HepG2 cells. POL showcased its capability to prevent HBV replication and effectively hinder viral entry, as evidenced by its IC50 values of 42.75 and 14.88 nM, respectively. In comparison, the standard anti-HBV drug, lamivudine (LAM), possessed an IC50 of 1.04 µM, yet did not manifest any blocking or neutralization effects.Fig. 3Inhibitory effect of lectin. (a) IC50 values of POL and SOF inhibitory effects on the HCV replication. (b) IC50 values of POL and LAM inhibitory effects on the HBV replication. Data are illustrated as mean ± SEM. Different letters indicate the significance at p