Major royal-jelly protein 2 and its isoform X1 are two novel safe inhibitors for hepatitis C and B viral entry and replication

Journal Pre-proof Major royal-jelly protein 2 and its isoform X1 are two novel safe inhibitors for hepatitis C and B viral entry and replication

Noha H. Habashy, Marwa M. Abu-Serie PII:

S0141-8130(19)35918-5

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.09.080

Reference:

BIOMAC 13325

To appear in:

International Journal of Biological Macromolecules

Received date:

28 July 2019

Revised date:

31 August 2019

Accepted date:

10 September 2019

Please cite this article as: N.H. Habashy and M.M. Abu-Serie, Major royal-jelly protein 2 and its isoform X1 are two novel safe inhibitors for hepatitis C and B viral entry and replication, International Journal of Biological Macromolecules(2019), https://doi.org/ 10.1016/j.ijbiomac.2019.09.080

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© 2019 Published by Elsevier.

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Major royal-jelly protein 2 and its isoform X1 are two novel safe inhibitors for hepatitis C and B viral entry and replication Noha H. Habashy a*, Marwa M. Abu-Serie b. a

Biochemistry Department, Faculty of Science, Alexandria University, Alexandria

21511, Egypt. Department of Medical Biotechnology, Genetic Engineering, and Biotechnology Re-

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search Institute, City for Scientific Research and Technology Applications (SRTA-

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City), New Borg EL-Arab 21934, Alexandria, Egypt.

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Corresponding author

(a) Noha H. Habashy: Biochemistry Department, Faculty of Science, Alexandria

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University, Alexandria 21511, Egypt. Tel: +20 1273431731; fax: 002(03) 3911794.

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E-mail address: [email protected], [email protected].

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Abstract Infections with HCV and HBV are serious worldwide health problems. Here, we report the anti-HCV and -HBV proficiency of Apis mellifera major royal-jelly protein (MRJP) 2 and its isoform X1. The efficiency of these proteins was evaluated in vitro and their safety was examined in vivo in comparison with Sofosbuvir (SOF) drug. Various in-silico methodologies were achieved for better understanding the antiviral mecha-

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nism of these MRJPs. Results proved their precluding ability to the viral receptors, CD81 and scavenger receptor class B type I (SR-B1). In addition, they targeted HCV-

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NS3/NS4A protease, HCV-NS5B polymerase, and HBV-polymerase (DNA-dependent

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DNA polymerase, and reverse transcriptase). Co-treatment with these proteins exerted

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different efficiencies toward CD81 and SR-B1 (synergistic), HBV-enzymes (antagonis-

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tic), and HCV-enzymes (either additive or synergistic). The studied proteins maximized their antiviral effect by their safety and superior potency to SOF. Collectively, these

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outcomes will shed the light on MRJP2 and its isoform X1 as two promising safeinhibitors for both HCV and HBV.

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Keywords: Major royal-jelly protein (MRJP) 2; MRJP2 isoform X1; viral inhibitors.

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1. Introduction Hepatitis C virus (HCV) and hepatitis B virus (HBV) are the most common etiologies for liver diseases in the world. Since, they are the main causes of liver fibrosis, cirrhosis and hepatocellular carcinoma "HCC" [1]. The prevalence rate of HCV is approximately 150-200 million patients, while this rate exceeds 280 million HBV patients worldwide [2]. The treatment target for HCV and HBV is to inhibit the replication, re-

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duce inflammation in the liver, reverse fibrosis and stop the progression of cirrhosis and HCC [3]. Since 2002, HCV has been treated with interferon-free direct-acting antivirals

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(IFN-free DAAs), which can cure HCV after 8-12 weeks. Despite the efficiency of

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these drugs, they have many side effects such as osteoporosis [4], cardiopathy [5], renal

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toxicity [6] and pulmonary hypertension [7]. Furthermore, these drugs make reactiva-

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tion for HBV in co-infected patients with both HCV and HBV [3]. On the other hand, the treatment strategies for HBV are through using of IFN/ pegylated IFN or nucleoside

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analogs or their combination for long-term. However, they can't remove HBV or cure the infection and the long-term intake of these drugs leads to potential renal toxicity in

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addition to drug resistance development [3]. Thus, the discovery of an effective and safe therapy for HCV and HBV is of great importance and the urgent need of the day. Royal-jelly (RJ) is one of the most important functional foods. It is a viscous milky substance secreted from the young worker bees mandibular and hypopharyngeal salivary glands. It has many essential components, including carbohydrates, proteins, lipids, vitamins, mineral salts, and other compounds. RJ has many pharmacological activities such as immunomodulatory, vasoactive, anticancer, antioxidant, and neurogenesis-enhancing properties [8]. The most important RJ protein components are the major RJ protein (MRJP) family; it encompasses eight glycoproteins "MRJP1-8, 49 to 87 kDa" [9]. We recently published a new method for purifying MRJP2 and its isoform X1

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Journal Pre-proof and approved theirs in vitro activity against hepatic injury and cancer [10]. As an extension of this work, this study was conducted to evaluate the antiviral activities of these two purified proteins. To date, the antiviral activities of RJ and its components are undefined. Therefore, the present study considered the first in evaluating the potential antiHCV and anti-HBV activities of MRJP2 and its isoform X1 alongside the possible antiviral effect of their combination. Furthermore, the acute toxicity of these proteins was evaluated in male Albino rats due to the curative effect of any compound and its ability

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to improve the health care will be maximized if it is safe. Sofosbuvir ("SOF" common

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name: Sovaldi), the most recent IFN-free DAA recommended to patients with chronic

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HCV, used here as a standard [11]. We select only one antiviral drug to appreciate the

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difference between the single antiviral activity of the currently approved drugs compared to the dual role of our novel therapy using MRJPs. Hence, the currently approved

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HCV drugs unable to cure HBV and vice versa.

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2.1. Chemicals

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2. Materials and methods

Polyacrylamide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ficoll-hypaque, carboxymethyl (CM)-Sephadex, polyethylene glycol (PEG) 8000, and anti-tetraspanin molecule (cluster of differentiation 81, CD81)-fluorescein isothiocyanate (FITC) antibody (clone M38) purified immunoglobulin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ammonium sulfate was supplied from Nentech Ltd (NTL, Brixworth, Northants, UK). Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) were obtained from Lonza (USA). SOF drug was supplied by Gilead Sciences, Ink (Foster, Ireland). Each tablet (1g) contains 400 mg SOF and other inactive ingredients. Protease inhibitor cocktail (PIC) and Gene JET RNA purification kit were obtained from Thermo Scientific, USA. Tag4

Journal Pre-proof lite receptor ligand binding assay kit was supplied from Cisbio Bioassays (New England Biolabs, Ipswich, MA, USA). SensoLyte® 490 HCV-Protease Assay Kit was purchased from Anaspec (San Jose, CA). The filter paper disks (FN 8) were purchased from Niederschlag, Germany. Tritiated thymidine-methyl-5'-triphosphate [3H-TTP] (specific activity, 107 Ci/mmol) was supplied from Amersham (Bucks, UK). Reverse transcriptase (RT) assay colorimetric kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), albu-

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Other chemicals were obtained with a high grade.

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min, cholesterol, and triglycerides (TG) kits were purchased from Biosystem, Spain.

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2.2. Purification of MRJP2 and MRJP2 isoform X1

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The fresh RJ was purchased from the local market of Egypt and used immediate-

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ly for purification of MRJP2 and MRJP2 X1 following our previous method [10]. Briefly, after discarding the non-soluble proteins, the crude protein fraction (CPF) and PF50

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were precipitated at 60% and (40-50 %) saturation of the ammonium sulfate, respective-

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ly. The two protein fractions (PFs) were dialyzed for 24 h against phosphate buffer saline (PBS) and quantified by Bradford method [12]. MRJP2 and its isoform X1 were purified from the prepared PF50 using CM-Sephadex ion-exchange column chromatography. The binding buffer was 20 mM phosphate buffer containing 1x PIC, pH 6.7 and the elution buffer was the binding buffer containing 0.5 M NaCl. The unbound protein that was firstly obtained by washing the column was the MRJP2 X1 and the bound one was MRJP2. The two PFs were dialyzed for 24 h against PBS (pH 7) and quantified by Bradford method [12]. Then they were analyzed by 14 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis "SDS-PAGE" using Coomassie brilliant blue R-250 [13]. All the collected fractions (CPF, PF50, MRJP2, and MRJP2 X1) were lyophilized (Telstar, Terrassa, Spain) and kept at -80 until analyzed. 5

Journal Pre-proof 2.3. Zeta potential analysis Zeta potential (surface charge) of MRJP2 and its isoform X1 was measured using a Nano ZS Zetasizer instrument (Malvern, UK) at 25°C. A concentration of 10 mg/L from each purified protein was freshly prepared prior to measurement in Milli-Q water and PBS containing 1x PIC, pH 6.7. Samples were filtered using 0.2 µm syringe filter to remove any impurities or aggregates to increase the quality of the results. Determination of zeta potential based on laser Doppler electrophoresis and data was given as the mean

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value of three samples; each of which was measured three times.

2.4. Preparation of viral host cells and infectious serum samples

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The peripheral blood mononuclear cells (PBMCs) were used here as HCV and HBV

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host cells. They were obtained by Ficoll-Hypaque density gradient centrifugation method as

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described previously [14]. In brief, the blood samples from ten healthy volunteers were diluted with an equal volume of PBS, carefully layered on Ficoll-Hypaque, and centrifuged at

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2000 rpm, 25°C for 30 min. Then the undisturbed PBMCs layer (interface) was carefully

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transferred out, washed, and suspended in 5 ml of RPMI-1640 medium containing 10% FBS then counted using trypan blue stain. Informed consent was acquired from all donors before their blood was used. The human blood was used obeying the Research Ethical Committee (REC) published by the National Health and Medical Research Council policies and the Ministry of Health and Population, Egypt. This research has been received permission from the Department of Medical Biotechnology (SRTA-City) and the Department of Biochemistry (Faculty of Science, Alexandria University). Serum samples were obtained from HCV or HBV infected Egyptian patients “A.R.” after agreement of the ethics committee. The HCV and HBV samples were positive for genotype 4a and D, respectively. The infected patients were neither under treatment prior to the study nor co-infected. The sera were kept at -80°C until use. 6

Journal Pre-proof 2.5. Cytotoxicity analysis The cytotoxicity of the tested compounds on PBMCs was performed using MTT assay [15]. The PBMCs (105 cells) were treated separately with RJ-PFs or SOF at serial dilutions for 72 h along with the untreated cells (negative control). The absorbance was recorded at 570 nm via an ELISA reader (BMG LabTech, Germany) and the % cell viability was determined. The concentration of each of the tested compounds that caused

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100% PBMCs viability (EC100, safe concentrations) was calculated.

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2.6. Evaluation of the Anti-HCV and anti-HBV activities of RJ-PFs The antiviral potentials of RJ-PFs (PF50, MRJP2, and MRJP2 X1) were evaluat-

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ed quantitatively using SOF for comparison. The viral host cells, human PBMCs (1x106

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cells) were seeded in each 12-well culture plate. For cell infection, plates were incubat-

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ed with either HCV (2.9 × 105 copies/ ml) or HBV (1 × 105 copies/ ml) infected serum in RPMI-1640 medium. The incubation was done in the CO2 incubator (New Brunswick

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Scientific, Netherlands) for 2 h at 37ºC, 5% CO2 and 95% humidity [16,17]. Then dif-

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ferent concentrations (1000, 500, 250, 0.1, 0.05, 0.025 µg) or (1000, 500, 250 µg) of PF50, MRJP2, and MRJP2 X1 were incubated with the HCV- or HBV-infected cells, respectively. SOF at a concentration of 4 mg was incubated only with HCV or HBVinfected PBMCs. All cells were incubated in the CO2 incubator for 72 h, and then the HCV-RNA and HBV-DNA within these cells were quantified using the fully automated Cobas Ampliprep Cobas TaqMan (CAP-CTM) analyzer (Roche Diagnostics, USA). 2.7. Antiviral mechanisms of MRJP2 and MRJP2 X1 2.7.1. Effect of MRJP2 and MRJP2 X1 on the early viral entry

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Journal Pre-proof The present study tested the ability of the RJ-PFs in prevention of the viral entry to host cells (PBMCs) using two mechanisms. The first was general (blocking and neutralization methods) and the second was specific (blockage of CD81 and SR-B1). The cellular blocking and viral inactivation (neutralization) assays were done following the previous method of Lin et al. with modifications [18]. In the cellular blocking assay, 1 mg of PF50, MRJP2 or MRJP2 X1 or 4 mg of SOF was incubated individually with the uninfected PBMCs for 2 h. After washing, the viral host cells were

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infected as described above with either HCV or HBV in RPMI-1640 medium for 2h. In

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the viral inactivation assay, the incubation was done between each of the PFs or SOF

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with the virus for 2 h before their incubation with the host cells for further 2h. Then in

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either two methods, the medium was discarded and replaced by fresh medium containing 10% FBS. Two control cultures were included and incubated under the same condi-

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tions, positive (infected cells only) and negative (uninfected cells). After 24 h incuba-

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tion, cells were washed and the viral load within the cells was quantified by CAP-CTM analyzer following the manufacturer's instructions [19].

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The blocking effect of the RJ-PFs on CD81 was investigated by the flow cytometry (Partec, Germany) using anti-CD81-FITC antibody. In brief, 90 µl (106 cells) of PBMCs were incubated for 2h with 1 mg of PF50, MRJP2, MRJP2 X1, or SOF. Then 10 µl of the antibody solution was added to the cell suspension and incubated at 4°C for 30 min. Afterward, the mixture was completed to 1 mL with PBS, centrifuged (2000 rpm, 5 min), and cells suspended in 2 ml PBS before being detected by the FITC signal detector (FL1). The blocking ability of the studied compounds to SR-B1 was examined using Tag-lite receptor-ligand binding assay following the manual protocol. This technology is a cellular screening platform that combines the homogeneous time-resolved fluores-

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Journal Pre-proof cence (HTRF) and SNAP-tag (self-labeling protein tag) technologies. The terbium (Tb) cryptate derivative (SNAP-Lumi4-Tb)-labeled SR-B1-expressed cells were mixed with the fluorescence-labeled SR-B1 ligand. Then different concentrations (10-1000 ng/mL) from each of the RJ-PFs or SOF were added and the fluorescence was measured at zero and 30 min using HTRF reader. The tag-lite technology is appropriate also for both saturation and competitive binding assays. The saturation binding method measures nonspecific and total binding of the fluorescent ligand at equilibrium then the specific bind-

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ing of the tested proteins was calculated as the difference. The concentration of protein

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occupying 50% of the total receptor binding sites (dissociation constant, Kd) was de-

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termined. While in the competitive binding assay, the tested compound was titrated into

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a solution containing a fixed amount of cells and a fixed concentration of fluorescent ligand to measure the inhibition constant (Ki). The values of Kd and Ki refer to the af-

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finity between the receptor and the labeled or unlabeled ligand, respectively.

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2.7.2. Effect of MRJP2 and MRJP2 X1 on HCV-non-structural (NS) protein

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3/NS4A protease and NS5B polymerase (POL) activities The inhibitory effect of RJ-PFs and SOF at different concentrations (10-1000 ng/mL) on HCV-NS3/NS4A protease was determined by SensoLyte® 490 HCVProtease Assay Kit following the manual protocol. This kit based on the incubation of labeled-fluorescence peptide substrate with a sequence derived from the cleavage site of NS4A/NS4B with the NS3/NS4A protease for 30 min at 37°C. Upon the proteolytic cleavage, the fluorescence was emitted and its intensity was measured at excitation/emission of 340 nm/490 nm using fluorescent phase contrast microscope (Olympus, Japan). While the inhibitory effect of different concentrations (10-40 ng/mL) of the studied compounds on NS5B POL activity was examined following the protocol of Ferrari et al. [20]. This protocol depends on the de novo synthesis of RNA by HCV-NS5B 9

Journal Pre-proof (genotype 1b-Con1)-Δ21-His 6 enzyme, which was expressed and purified according to Ferrari et al. method [21]. The enzyme used heteropolymeric modified (at its 3′-end with dideoxycytidine) RNA template and radiolabeled nucleotide. The incorporation of the radiolabeled nucleotide during the enzyme's de novo initiation and elongation cycles was read using the TopCount NXT microplate scintillation counter (Perkin Elmer, Wellesley, MA). In both enzymatic assays, the control reaction (maximum enzyme activity) was performed using the enzyme without any of the compounds being studied.

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The inhibition rate of both enzymes was expressed as % of inhibition for calculation of

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the IC50 values (the concentration of the tested compound that caused 50% enzyme in-

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hibition) from the dose-response curve.

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POL (DDDP) and RT activities

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2.7.3. Effect of MRJP2 and MRJP2 X1on the HBV-POL, DNA-dependent DNA

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The inhibitory capacity of RJ-PFs (10-1000 ng/mL) on HBV-DDDP activity compared to SOF was achieved using the modified method of Hirschman et al. [22]. In

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brief, the enzymatic reaction (49 µl) contained a mixture of 21 µl of virus suspension, 7 µl of each studied protein or SOF, 340 mM KCl, 34 mM MgCl2, and 0.4% Nonidet P40. In addition, 42 mM Tris-HCl (pH 7.5), 22 mM β-mercaptoethanol, and 70 µM each of the required deoxynucleotides including 1µCi radiolabeled [3H-TTP] were included. Then the newly synthesized DNA was transferred to the filter paper disks and was precipitated using 5% trichloroacetic acid to measure the radioactivity using the scintillation counter. The HBV-RT assay was performed using the RT assay colorimetric kit following the manufacturer’s instructions. In brief, the HBV-infected PBMCs were incubated separately with different concentrations (10-1000 ng/mL) of PF50, MRJP2, MRJP2 X1,

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Journal Pre-proof or SOF for 2h. Then the HBV particles were precipitated from the culture medium using PEG 8000 (30%) for 12 h at 4°C and suspended in lysis buffer for 30 min at 25°C. The RT enzymatic reaction was started by adding the substrate mixture [the template/primer hybrid poly (A) × oligo (dt)15 and labeled nucleotides with digoxigenin and biotin in an optimized ratio] for the DNA synthesis. The detection and quantification of the synthesized DNA molecule follow a sandwich ELISA protocol. Two controls were included, the negative control (without the virus lysate) and the positive control (without the test-

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ed compounds). The percentage inhibition and IC50 values of the studied compounds

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were calculated.

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2.8. Computational analyses

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The present study developed different computational methods (in silico assess-

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ments) to analyze the physical properties and the inhibitory mechanisms of MRJP2 and MRJP2 X1 on viral entry and replication.

values

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the

protein

amino

acids

via

the

EXPASY

server

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pK

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The theoretical isoelectric point (PI) of the two MRJPs was predicted from the

"https://web.expasy.org/compute_pi/" [23]. The I-TASSER (Iterative Threading ASSEmbly Refinement) server "http://zhanglab.ccmb.med.umich.edu/I-TASSER/" [24] predict the 3D protein structures that had no available structural templates in the Protein Data Bank (PDB, https://www.rcsb.org/). These proteins include the two tested Apis mellifera proteins, MRJP2 (Accession: ACS66837, 452 amino acids) and MRJP2 X1 (Accession:

XP_026299315, 452 amino acids). In addition, SR-B1 (Accession:

NG_028199, 509 amino acids) and HBV-POL (Accession: AGA95798, 843 amino acids) were comprised. The HBV-POL structure is divided into four regions, terminal protein (TP) domain (1-352), spacer domain (353-515), DDDP/RT (T) domain (516-600), and RNase H (RH) domain (601-843). The amino acid sequence of these proteins was 11

Journal Pre-proof provided from the NCBI (https://www.ncbi.nlm.nih.gov/protein/) protein database and then submitted to the I-TASSER server to predict their 3D structure. This can be achieved by aligning the protein sequence with the PDB structural protein templates (threading technique) to construct five full-length structure models by iterative fragment assembly simulations. From these models, the one with the highest C-score value signifies a high confidence model and then it was matched in the PDB library to all protein structures to know the structural analogs by TM-align program.

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The COFACTOR "https://zhanglab.ccmb.med.umich.edu/COFACTOR/" [25]

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and COACH "https://zhanglab.ccmb.med.umich.edu/COACH/" [26] servers were used

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to predict the protein function and protein-ligand binding site based on its structure.

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For a better understanding of the inhibitory roles of the MRJP2 and MRJP2 X1 on early viral entry and replication, protein-protein docking was investigated. Docking

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method was carried out using GRAMM-X Protein-Protein Docking Web Server [27].

The

obtained

I-

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"http://vakser.compbio.ku.edu/resources/gramm/grammx/"

TASSER model 1 for MRJP2 or MRJP2 X1 was docked with CD81 (PDB ID: 1G8Q),

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HCV-NS3/NS4A (PDB ID: 1NS3), or HCV-NS5B POL (PDB ID: 3MWV). In addition, the I-TASSER model 1 of SR-B1 and HBV-POL has docked individually with these studied RJ-purified proteins. Then the docked protein complexes were analyzed by PIMA (Protein-Protein Interactions in Macromolecular Assemblies) server "http://caps.ncbs.res.in/pima/" [28]. This tool investigated the interchain interactions in a docked complex, including interface residues, van der Waals pairs, salt bridge, hydrophobic, favorable and unfavorable electrostatic interactions. In addition, this tool provided the total number of atoms in each chain in the protein complex. The docked protein complex was visualized and further analyzed by the Discovery Studio 2017 R2 program to elucidate the interacting residues in the binding sites.

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Journal Pre-proof 2.9. Assessment of PF50 and SOF acute toxicity Karber's method [29] had been used to study the acute toxicity effect of PF50 compared to SOF using male Albino rats and the median lethal dosage (LD50) was calculated. 2.9.1. Experimental animals and design Fifty-five male Albino rats (80-120 g) were purchased from MISR University for Science and Technology (animal welfare insurance number A5865-01). Animals

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were housed in metal cages and allowed free access to a standard commercial diet and

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tap water. Rats were kept under conventional humidity, temperature, and twelve hours

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light/dark cycle. All rats were acclimatized for one week prior to handling in the labora-

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tory environment and were daily observed for abnormal signs. Handling of animals complied with the guidelines of the Research Ethical Committee in Egypt.

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Animals were divided into ten groups (five animals in each) starting from group

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"A" to group "J" beside the control, which had no treatment. Groups from "A" to "E" received PF50 intraperitoneally, while groups from "F" to "J" received SOF orally. PF50

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or SOF were administered to animals at different doses (140, 350, 700, 2500 and 5000 mg/kg body weight "bw"), one dose per group of animals (group "A" and "F" received the lowest dose).

2.9.2. Hematological, biochemical, and histopathological studies All animals were left for 48 h and then the mortality rate was recorded in each group for the LD50 value calculation. Also, the weights of animals in each group were recorded before treatment and immediately before sacrifice. Blood samples were collected in heparin tubes by cardiac puncture for assessment of the erythrocytes, hematocrit, hematimetric indices, hemoglobin (Hb), platelets, and leukocytes (full and differential counts). In addition, routine liver and kidney function parameters, as well as TG and 13

Journal Pre-proof cholesterol levels, were determined using specific kits. The vital organs including liver, lung, kidney, and spleen were carefully excised, weighed, and examined morphologically using hematoxylin and eosin (H & E) stain. 2.10. Synergy testing The synergy testing has been studied to evaluate the effect of the MRJP2 and MRJP2 X1 combination as PF50 on the antiviral potency using the combination index

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(CI) analysis. The CompuSyn software, one of the modern tools used for calculating the

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CI value and plot based on Chou and Talalay median-effect analyses [30], is used here with the viral enzymes and SR-B1 results. However, the CI values for the CD81 result

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were calculated by dividing the expectable value on the practical value. The expectable

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value between MRJP2 and MRJP2 X1 was calculated from the formula: [(practical val-

2.11. Data analysis

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(control value) [31].

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ue for MRJP2)/(control value)] X [(practical value for MRJP2 X1)/(control value)] X

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Data were expressed as mean ± SE and analyzed by SPSS version 16. The mean values were compared using one-way analysis of variance (ANOVA) by Duncan's test and the significance was determined at P < 0.05. IC50 and EC100 values were calculated by the GraphPad Instat software version 3 and the ClustVis web tool designed a heat map diagram "https://biit.cs.ut.ee/clustvis/" [32]. The CompuSyn software (ComboSyn, Inc, Paramus, NJ) accomplished the CI values and plots for viral enzymes and SR-B1 results. 3. Results 3.1. Purification of MRJP2 and MRJP2 X1and their properties

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Journal Pre-proof The total water-soluble proteins (CPF) in RJ were precipitated at 60% ammonium sulfate saturation yielding 14.30 ± 0.01 g%. While the PF50 was obtained at the yield of 2.55±0.01 g% and contained nearly equal amounts of MRJP2 (1.22±0.02 g%) and MRJP2 X1 (1.13±0.03 g%). The SDS-PAGE analysis (Fig. 1I) elucidated that the RJ-CPF gave fourteen protein bands with an estimated molecular weight range of 10.81-100.00 kDa. In addition, the PF50 revealed two protein bands of 45.41 and 50.55 kDa. However, only one distinc-

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tive band was detected for each of the two purified proteins (MRJP2 and MRJP2 X1)

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with a molecular weight of 49.5 and 53.12 kDa, respectively. The zeta potential results

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of MRJP2 and MRJP2 X1 in milli Q water (Fig. 1IV and V) showed a single peak for

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each protein. Moreover, both proteins carry a negative net charge with the value of 11.1 and -14.1, respectively in water and -7.11 and -11.3, respectively in PBS (pH 6.7).

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The RJ purified proteins are monomers with three domains and each contains 452

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amino acids as provided by the Vector Alignment Search Tool (VAST) and the NCBI database information, respectively. The predicted PI values for the two proteins as

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achieved by the EXPASY server are slightly different. Hence, MRJP2 showed a PI value of 6.8 and MRJP2 X1 had the PI value of 6.6. The 3D structures of MRJP2 and MRJP2 X1 were predicted using the I-TASSER in silico analysis, the most successful method of predicting protein structure. Highest C-score value models are the highest quality 3D structures that used here for both proteins (Fig. 1II and III). COFACTOR and COACH analyses predicted certain molecular, cellular, and biological functions for MRJP2 and its isoform X1 based on their I-TASSER 3D structures. Some of these predicted functions included receptor activity, cell-matrix adhesion, extracellular-matrix proteins binding, and receptor/integrin complex. 3.2. Effect of the RJ-PFs on the host cell viability 15

Journal Pre-proof The cytotoxic effect of RJ-PFs and SOF was checked on the viral host cells, (PBMCs). The results revealed that incubation of PF50, MRJP2, and MRJP2 X1 with the PBMCs had no cytotoxic effects with safe concentrations (EC100 mg/mL) values of 2.942±0.045, 2.584±0.258, and 3.311±0.262, respectively. The MRJP2 X1 was significantly (P ˂ 0.05) safer than the MRJP2 and PF50, but SOF was significantly (P ˂ 0.05) less safe than the MRJPs on the PBMCs and had an EC100 value of 0.193±0.003.

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3.3. Antiviral efficiency of RJ-PFs

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The hepatitis C and B viral loads were precisely quantified in PBMCs cells using the fully automated CAP-CTM analyzer. Results showed that both viral infections be-

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came undetectable after the treatment with the RJ-PFs at most of the studied doses.

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Treatment of HCV-infected PBMCs with all tested doses of PF50 (1000-0.025 µg) was

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able to clear the total viral load inside the cells (284,000 ± 0.01 IU / mL, positive control). In addition, MRJP2 and MRJP2 X1 were able to clear the HCV inside the PBMCs

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at all the tested doses, except at 0.025 µg, only about 1000 ± 0.01 IU / mL of HCV re-

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mained. The SOF at the dose of 4 mg could not inhibit all the viral infection (324 ± 0.02 IU/mL remained).

Concerning HBV-infected PBMCs, a very low detectable viral load (around 600 ± 0.03 IU/ mL) was observed only at 250 µg of MRJP2 or MRJP2 X1 from the total viral load of 110,000 ± 0.01 IU/mL. In contrast, SOF showed no effect on HBV infection. 3.4. Blocking of the viral host cell receptors by RJ-PFs The incubation of HCV or HBV with untreated PBMCs for 2h (positive control) culminated in the entry of 4,800 ± 0.02 IU/mL of HCV and 37,500 ± 0.03 IU/mL of HBV into the cells. Treatment with 1 mg of PF50 or MRJP2 prevents the entry of HCV and HBV into the host cells in the blocking method. However, the same concentration

16

Journal Pre-proof of MRJP2 X1 blocks the entry of most HCV and HBV viral loads with the exception of about 24 ± 0.05 IU/mL of both viruses. On the other hand, none of these PFs can preclude the entry of HCV or HBV into host cells in the neutralization method and viral loads nearly equal the positive control values. The ability of SOF to block the HCV and HBV entry demonstrated that the dose of 4 mg prevented most of the HCV entry into PBMCs, with the exception of 963 ± 0.00 IU/mL in the blocking method. However, it could not prevent the entry of either HCV in the neutralization method or HBV.

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In addition to the previous general mechanisms for viral entry, we tested the

ro

blocking effect of RJ-PFs on HCV cellular receptors (CD81 and SR-B1). The flow cy-

-p

tometric investigation of CD81 using the FITC-anti-CD81 antibody demonstrated the

re

expression of this receptor on the cell surface of PBMCs (Fig. 2I and II). The incubation of RJ-PFs with PBMCs was markedly reduced levels of unbound CD81 (i.e. increased

lP

binding to the receptor) by varying extents. The highest binding capability was detected

na

with PF50 followed by MRJP2 then MRJP2 X1. The calculated CI value for binding PF50 to CD81 on the cell surface of the PBMCs was 0.458. The predicting analysis re-

Jo ur

vealed different types of weak interactions between MRJP2 or MRJP2 X1 to CD81 receptor chain A (Fig. 2III-VI). In comparison with SOF, RJ-PFs showed a significant (P ˂ 0.05) higher binding capability to CD81. The results also demonstrated the binding of MRJP2 and MRJP2 X1 to SR-B1 receptor in a concentration-dependent manner (Fig. 3I). The IC50 values revealed the highest potency (the lowest IC50 value) of MRJP2 X1 in SR-B1 binding followed by PF50 then MRJP2 (Fig. 3III). In contrast, SOF was unable to block the SR-B1 at the studied concentrations (10-1000 ng/mL). The CI plot for PF50 (Fig. 3II) was constructed by the CompuSyn software from the calculated CI values at each studied concentration (10-1000 ng/mL) of PF50. The results revealed that the CI values were ˂ 1 at all these

17

Journal Pre-proof tested concentrations. Equilibrium competition- and saturation-binding assays were accomplished to know the affinity and efficiency of the purified proteins to the SR-B1. As shown in Fig. 3IV the Kd value of MRJP2 significantly (P ˂ 0.05) higher (lower affinity) than MRJP2 X1 (higher affinity) while both proteins had two closely related Ki values. As seen in Fig. 3V-VIII, each of the two purified MRJPs binds to SR-B1 through different types of non-covalent interactions and the number of atoms in each chain in

of

the docked complexes was provided.

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3.5. Effect of RJ-PFs on HCV and HBV replication

The influence of RJ-PFs on HCV protease (NS3/NS4A), HCV-NS5B POL, and

-p

HBV-POL (DDDP and RT activity) was evaluated here and compared with SOF. The

re

results demonstrated concentration-dependent inhibition of the activities of these repli-

lP

cation enzymes (Fig. 4I, II, IV and V). The IC50 values (Fig. 4VI) elucidated the potency of this inhibition and heat map plots clustered these results.

na

With respect to the HCV protease, the results revealed the closely related (non-

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significant difference in IC50 values) inhibitory efficiency of PF50 and MRJP2 which was the same as SOF. However, MRJP2 X1 exhibited the lowest inhibitory potency for the HCV protease. On the other hand, different efficiencies of the RJ-PFs towards HCV-NS5B were observed. Hence, the MRJP2 exhibited the highest inhibitory potency followed by PF50 and MRJP2 X1, while the lowest inhibitory effect was shown by SOF. With regard to the Chou and Talalay CI values of PF50, the majority of these calculated values for HCV-NS3/NS4A and a half for HCV-NS5B were less than one (Fig. 4VIII). Concerning the HBV-DDDP, MRJP2 had the lowest IC50 value, followed by PF50 and MRJP2 X1. However, slight differences were observed in the IC50 values of the studied RJ-PFs against HBV-RT (Fig. 4VI). In contrast, SOF didn't reveal any effect

18

Journal Pre-proof on HBV-DDDP and -RT activities at all concentrations studied. The calculated CI values for PF50 were more than one for both HBV catalytic domains (Fig. 4VIII). The heat map plot (Fig. 4VII) summarizes the inhibitory efficiency of RJ-PFs for all the studied HCV and HBV replication enzymes. While the heat map diagram in Fig. 4III showed a comparison between the three RJ-PFs and SOF on HCV replication enzymes.

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3.6. Viral enzymes docking and interaction analyses

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To identify the binding mode of MRJP2 and its isoform X1 to HCV and HBV replication enzymes that involved in their inhibitory role, the docked enzyme-MRJP

-p

complex was built and then analyzed by PIMA web server. Results revealed that

re

MRJP2 or MRJP2 X1 can interact with chain A and B of the NS3 protease and chain C

lP

only or chain C and D of the NS4A peptide, respectively. However, both MRJPs can interact only with chain A of the dimeric protein HCV-NS5B POL. Fig. (5I-VI) and (6I-

na

VI) identified the different types of non-covalent interactions, the number of interacting

Jo ur

residues, and the interacting chains of HCV-NS3/NS4A and -NS5B, respectively. The present study used the COFACTOR web server to predict the viral enzymes active sites when aligning the chain A of each enzyme with the templates in the protein function databases. Only the top five homologous enzymes were used to predict catalytic sites. Data showed that some of the predicted active site residues in the HCV-NS5B POL chain A were involved in the interaction with the tested proteins. Hence, MRJP2 can interact with K51, D220, D319, S367, and V370, but MRJP2 X1 binds only to K51 (Fig. 6III and V). However, none of the predicted active site residues of the HCVNS3/NS4A protease contributed to the interaction with MRJP2 or MRJP2 X1. Fig. (7IVI) demonstrated the outputs of the HBV-POL docking and interaction characterization.

19

Journal Pre-proof The prediction of the HBV-POL active sites by COFACTOR web server gave no results and in turn, it needs further investigations. 3.7. Acute toxicity study The results of PF50 or SOF administration at different doses for 48 h showed no abnormal behavioral changes, no significant body weight gain or loss, and no mortality in the studied groups. The LD50 value of both PF50 and SOF is more than 5000 mg/kg

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bw.

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The organ-to-body weight ratio data showed no significant difference from the control in the animals received various doses of PF50 (Fig. 8I). However, animals ad-

-p

ministered SOF showed abnormal changes in the weight and appearance of the organs,

re

hence they looked larger than the control at the different studied doses (Fig. 8II). In

lP

harmony with these results, the histopathological examination of the liver, kidney, and spleen sections (Fig. 8III) in the rats dosed with 140 and 5000 mg/kg bw of PF50 (group

na

B, E, respectively) did not reveal any morphological changes. Only a slight thickening

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of the alveolar septae was observed in the lung section of both doses. However, intake of SOF caused morphological abnormalities and disturbance in the architecture of all examined organ sections. Hence, the administration of low-dose (140 mg/kg bw, group F) resulted in slight changes in liver architecture as well as dilation in the renal tubules with glomerular damage. In addition, the lung showed thickening in the alveolar septae while the spleen revealed normal architecture. However, high-dose administration of SOF (5000 mg/kg bw, group J) resulted in hepatic sinusoidal dilation, hepatocyte vacuolation and swelling (necrosis), and bile duct proliferation. Moreover, severe renal tubules dilation and glomerular damage; increased alveolar septae thickening with narrowing of their spaces, as well as infiltration of inflammatory cells in the spleen was detected. 20

Journal Pre-proof The results found slight changes in some hematological indices (HCT "hematocrite", MCV "mean corpuscular volume", MCH "mean corpuscular Hb", granulocytes) in rats administered PF50 at certain concentrations (Table 1). However, the intake of SOF at different concentrations (Table 2) resulted in a significant (P ˂ 0.05) increase in total white blood cells (WBCs), granulocytes, and monocytes and a significant drop in platelets. With regard to the biochemical studies, the injection of PF50 into rats didn't cause any abnormal changes in the studied parameters compared to the control group

of

(Table 1). Interestingly, intake of PF50 at and above 140 mg/kg bw obviously reduced

ro

sodium, TG, and cholesterol levels. In contrast, oral administration of SOF revealed ab-

-p

normal liver and kidney function parameters (Table 2) that reflected the liver and kid-

re

ney morphological damage. At the higher dose (5000 mg/kg bw) of SOF, the levels of AST, urea, sodium, TG, and cholesterol were significantly high (P ˂ 0.05) compared to

lP

control. Some of these parameters were raised at doses below 5000 mg/kg bw. In addi-

na

tion, the level of albumin and potassium was significantly (P ˂ 0.05) reduced after ad-

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ministration of the higher dose of SOF.

4. Discussion

The HCV and HBV are the most public types of hepatitis and are potentially fatal diseases. They are treated with some synthetic drugs with massive side effects [5–7], so novel safe drugs are urgently needed. The current work identified a new therapeutic strategy for HCV and HBV using MRJP2 and its isoform X1. The two proteins have been purified using our recently published method of ammonium sulfate precipitation (at 40-50% saturation) followed by ion exchange chromatographic separation [10]. The success of the separation was confirmed by the appearance of single band purity for each protein on the SDS-PAGE gel with closely related molecular weights. The MRJP2 showed the molecular weight of 49.50 kDa and 53.12 kDa was reported for MRJP2 X1. 21

Journal Pre-proof This result was in line with the previous study of Schmitzova et al. [33]. In addition, the zeta potential of the two purified proteins was determined and their negatively charged value (Fig. 1IV andV) reflected the anionic properties of the two MRJPs. Also, the single peak for each protein confirmed the presence of such a protein in a pure state. The results of this study demonstrated the change in the value of zeta potential for both MRJPs following the slight change in solvent pH. Hence, these values in milli Q water (pH 7) were more negative than in PBS (pH 6.7). This may be due to the decrease in net

of

positive charges on the surface of each studied protein with raising the pH, and these

ro

data are agreed with the previous studies [34]. Our previous study on MRJP2 and

-p

MRJP2 X1 showed that the PI values of these two proteins are 7 and 6.5, respectively,

re

as obtained theoretically by the MALDI-TOF analysis [10]. Also, the predicted PI values for the two proteins from their amino acid sequences using the EXPASY server

lP

were nearly the same (6.8 and 6.6, respectively). From the PI values, we can speculate

na

the net charges on these proteins at pH 6.7 (The pH used in purification) as a net positive for MRJP2 and a net negative for MRJP2 X1. However, the actual surface charges

Jo ur

of the two proteins were different and, more surprisingly, virtually more negative than expected from sequence alone (Fig. 1IV and V). This proposes the occurrence of posttranslational charge modifications for the two proteins, which may be attributed to glycosylation. Hence MRJP2 [35] and other MRJPs [36] were reported previously as extremely glycosylated proteins. Prolongation of the newly published work on the dual anti-hepatic injury and cancer role of MRJP2 and MRJP2 X1, this study evaluated the antiviral potential of these two proteins separately or together (PF50). Since there is a limited host range for HCV and HBV and the newly established in vivo models for these two viruses have high costs and inadequate outputs. The cell culture-based systems are therefore fre-

22

Journal Pre-proof quently used to evaluate newly discovered drugs [37]. Thus, this study evaluated the in vitro antiviral role of the two MRJPs using PBMCs. Although HCV and HBV are hepatotropic viruses and their main target is hepatocytes, there is strong evidence to confirm their replication in extrahepatic reservoirs such as PBMCs [38–40]. The results confirmed the safety of MRJPs and PF50 on PBMCs, which is significantly greater (higher EC100) than SOF. In this study, the MRJP2 and its isoform X1 have been identified as potent anti-

of

viral proteins against both HCV and HBV, indicating the dual antiviral potency of these

ro

two studied MRJPs. The results suggested that the viral inhibitory efficacy of these pro-

-p

teins was dose-dependent and that the minimum effective dose of these proteins was

re

0.05 µg for HCV and 500 μg for HBV with higher potency to MRJP2. On the other hand, SOF at 4 mg cleared some of HCV infection and was unable to prevent HBV in-

lP

fection under the same conditions. Therefore, it is clear that the two studied MRJPs had

proved drug, SOF.

na

strong dual viral inhibitory activity with highly greater efficiency than the recently ap-

Jo ur

The data showed that these proteins had multiple antiviral mechanisms of action. Their influence is established predominantly on the inhibition of two critical stages during the viral life cycle, the initial stage of entry and genomic expression. This can be happened by targeting the host cell receptors and the viral replication enzymes, respectively, but they cannot bind directly to the virus itself. Blocking cellular CD81 and SRB1 receptors by these proteins (Fig. 2 and 3) conferring host resistance to viral infection. From the docking analysis, we observed the ability of these MRJPs to bind with the extracellular domain (chain A) of CD81 or SR-B1 by different types of weakinteractions (Fig. 2IV and VI, 3V and VI). Moreover, there is an obvious synergistic (CI ˂ 1) binding of PF50 (MRJP2 and MRJP2 X1 combined PF) to each of these two

23

Journal Pre-proof receptors. These results were in line with the predicted function obtained from the COFACTOR server on the ability of these MRJPs to bind with the extracellular matrix proteins. The results showed a higher efficiency of MRJP2 in binding to CD81, whereas MRJP2 X1 had a higher binding affinity (lower Kd value) to SR-B1. However, the overall ability of MRJP2 to prevent the entry of HCV and HBV into either host cell was superior to MRJP2 X1. In contrast, SOF exhibited less potency in binding to CD81 and was unable to block the SR-B1 receptor or prevent HBV entry. Accordingly, the highest

of

antiviral efficiency of MRJP2 may be attributed. CD81 was the first identified HCV cel-

ro

lular receptor capable of binding to the envelope (E)2 protein virion [41]. Similarly, SR-

-p

B1 facilitates the HCV virion binding to the host cell by interacting with its associated

re

lipoproteins and E2 protein. However, the only recognized HBV receptors are the sodium taurocholate co-transporting polypeptide (NTCP) and heparan sulfate proteoglycans

lP

(HSPG) that attach to the HBV preS1 surface protein. The CD81 and SR-B1 are located

na

on the surface of PBMCs [42]. Therefore, the blocking effect of the two MRJPs to these two receptors can be considered as one mechanism to prevent the entry of HCV into

Jo ur

PBMCs. The NTCP is not expressed on the PBMCs surface [43], but specific proteoglycans like glypican (GPC) are expressed and may be involved in the HBV entry [44]. However, the exact mechanism of HBV entry into lymphatic cells has not yet been identified and would like to be explored [38]. From the previous outcomes, we can consider MRJP2 and its isoform as HCV and HBV entry inhibitors. Therefore, they can prohibit naive cell and cell-to-cell entry, which is particularly significant for liver transplantation and viral transmission from mothers to children. Viral entry inhibitors are now urgently needed to overcome resistance to the recently used antiviral drugs. Thus, MRJP2 and its isoform X1 are two promising novel antiviral compounds to avoid and treat infections with HCV and HBV.

24

Journal Pre-proof The NS3/NS4A and NS5B are two essential enzymes for HCV replication by monitoring the viral genomic expression. Hence, NS3 has a helicase/ATPase activity in its C-terminal portion to unwind and bind the viral RNA genome and has both NTPase and serine protease activity in its N-terminal portion. This enzyme required NS4A as a co-factor and the enzyme complex (NS3/NS4A) contributes to HCV pathogenesis. While NS5B (RNA-dependent RNA POL) is essential in initiating the synthesis of the viral RNA negative strand. This enzyme has no proofreading function so that different

of

mutants are created during transcription leading to drug resistance. Owed to the key role

ro

of both NS3/NS4A and NS5B in the HCV replication, they are considered as prospec-

-p

tive targets for anti-HCV drugs [45]. While HBV replication is based primarily on

re

HBV-POL, the only viral protein with enzymatic function. This enzyme comprises three functional domains: (from C-terminal to N-terminal) RH, T, and TP domain. It partici-

lP

pates in multiple events of the viral replication cycle, including encapsidation (or pack-

na

aging), RNA binding, template switching, RNA protein priming, RNA degradation, and

drugs [46].

Jo ur

DNA synthesis. Therefore, this enzyme serves as the main target for the anti-HBV

The MRJP2 and its isoform X1 showed a powerful ability to inhibit the activity of the above essential HCV and HBV enzymes and this inhibition was concentration dependent. In addition, PF50 (Fig. 4VIII) inhibited the activity of NS3/NS4A synergistically (CI ˂ 1), NS5B additively (CI = 1), and both HBV POL catalytic domains antagonistically (CI ˃ 1). The IC50 value of the HCV and HBV replication enzymes (Fig. 4VI and VII) revealed the highest inhibitory effect of MRJP2. The PIMA computational results revealed different types of interaction between MRJP2 or MRJP2 X1 and these viral replication enzymes (Fig. 5-7). In addition, the COFACTOR computational findings demonstrated the ability of these two proteins to interact with the predicted active

25

Journal Pre-proof site residues of HCV-NS5B POL but not with HCV-NS3/NS4A protease. We can, therefore, conclude the inhibition mode of these proteins based on the previous literature (Sharma, 2012) as competitive inhibition to HCV-NS5B POL and uncompetitive or non-competitive inhibition to HCV-NS3/NS4A protease. Thus, these proteins have the same mechanism of action as some of the recently approved drugs as boceprevir, telaprevir [45], and SOF [49]. While after comparing the efficiency with SOF, MRJP2 and PF50 exhibited similar inhibitory effect for HCV-NS3/NS4A protease and higher

of

inhibitory effect for HCV-NS5B POL. In contrast, MRJP2 X1 showed lower potency in

ro

the inhibition of HCV-protease activity and higher efficiency in the suppression of

-p

HCV-NS5B activity. Consequently, these MRJPs, especially MRJP2 might be consid-

re

ered in the strategy of the new anti-HCV drugs.

With respect to HBV-POL, MRJP2 and its isoform X1 were predicted from the

lP

docking analysis to bind in different regions of the enzyme. Hence, MRJP2 was pre-

na

sumed to bind only to the TP domain, but MRJP2 X1 can interact with all the enzyme domains at certain amino acid residues (Fig. 7). However, the two proteins revealed

Jo ur

their inhibitory efficiency to both DDDP and RT activities of HBV-POL (Fig. 4IV and V). Therefore, one of the anti-HBV mechanisms of these proteins was similar to many newly approved HBV drugs such as Lamivudine, Telbivudine and Adefovir [50,51]. The COFACTOR analysis of HBV-POL showed no active sites prediction, which may be due to this enzyme containing certain distinctive structures such as the TP and spacer domains with no structural homologs [46]. Thus, we cannot predict the type of inhibition of the studied proteins to HBV-POL and this will need more examination. Although we can postulate that MRJP2 may inhibit the activity of HBV-DDDP or RT non or un-competitively because it does not bind to this domain. Nevertheless, by one or more reversible types of inhibition, competitive, non-competitive, or non-competitive,

26

Journal Pre-proof MRJP2 X1 may inhibit the activity of these enzymes. In contrast, SOF had no effect on the activity of HBV POL enzymatic domains (Fig. 4IV and V). However, no previous work disclosed the impact of SOF on the activity of HBV-POL, DDDP and RT domains. Just the ability of SOF to raise the level of HBV-DNA in HCV/HBV co-infected patients published in many articles [52]. The acute toxicity of MRJPs has been studied in the current work to assess the

of

safety of these new treatments. Hence novel drug development must be not only effec-

ro

tive but also safe or at least with minimal side effects to achieve the best therapeutic outcome. Single intraperitoneal administration of these proteins (PF50) to rats at various

-p

doses was achieved following Karber's method [29]. The results showed no mortality,

re

no abnormal weight gain or loss, and no abnormal signs with all the studied doses, even

lP

the highest (5000 mg/kg bw). In addition, the LD50 value (a common acute toxicity dose assessment) of PF50 is above 5000 mg/kg bw. This outcome is concordant with the

na

previous study on the acute and subacute toxicity of RJ [53]. The results also revealed

Jo ur

no change in the morphological appearance of the experimental animals' vital organs such as liver, kidney, and spleen, except for minor changes in lung morphology (Fig. 8III). In line with these findings, the safety of RJ oral administration in the liver and kidney for three months was confirmed [54]. The systemic toxicity that was evaluated here using biochemical tests (Table 1) with particular attention to liver and kidney function, showing no toxic effects. Interestingly, the administration of PF50 drastically reduced the levels of sodium, TG, and cholesterol. This may be due to the regulatory role of RJ in the angiotensin I-converting enzyme and sex hormones as reported previously [54,55]. Correspondingly, PF50 can be a valuable drug for patients with hypertension and cardiovascular diseases besides its antiviral potential. On the other hand, the administration of SOF generated toxicity in rats. This was manifested by an abnormal increase 27

Journal Pre-proof in the organ-body weight ratio and morphological damage of the examined organs, especially with the highest dose (5000 mg/kg bw) (Fig. 8). This disorder resulted in abnormal markers of liver and kidney function along with abnormal lipid and hematological profile (Table 2). Despite these disturbances, the LD50 of SOF was more than 5000 mg/kg bw due to no mortality occurred with all of its administered doses. The liver [56], lung [7], and kidney toxicity [6] of SOF have been confirmed before. Furthermore, the treatment with SOF caused retinopathy, uveitis [57], cardiopathy [5] as well as other

of

hematological side effects [58]. From all these findings, the present study revealed the

-p

verified the previously reported toxicity of SOF.

ro

safety of MRJPs on the experimental animals, even at high dose (5000 mg/kg bw) and

re

In summary, this study discovered new treatments for two life-threatening virus-

lP

es, HCV and HBV, naturally synthesized in the hypopharyngeal and maxillary glands of worker bees, MRJP2 and MRJP2 isoform X1. These proteins, especially MRJP2,

na

proved their super-efficacy against both HCV and HBV in vitro. In addition, these pro-

Jo ur

teins can combat the two main viral adverse effects, hepatic injury and cancer as reported before. Thus, they achieve the ultimate goal of developing a cure for HCV and HBV infection. The two proteins act principally by inhibiting the viral entry in the initial stage as well as vital replication enzymes. In addition, they are safe on the experimental animals and have superior effectiveness to SOF in all the studied items. Thus, these proteins are a starting point for the development of effective antiviral drugs and will revolutionize the field.

28

Journal Pre-proof Conflict of interest Authors have filed a Patent Cooperation Treaty application (international application number: PCT/EG2017/000022, Publication Date: August 2018) regarding the results in the current paper. This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors' contributions

ro

M.M.A and N.H.H contributed equally in conducting and designing the experi-

-p

ments; analyzing and interpreting data as well as writing and revising the manuscript.

re

Acknowledgments

lP

We are grateful to Mr. Salem E. El-Fiky for helping, encouraging, and providing us with the RJ to perform this work. Warm greetings to Dr. Esam R.Ahmed, Head of

na

Confirmatory Diagnostic Unit, Vacsera, Giza, Egypt, for evaluating the methodology

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Journal Pre-proof

Figure Legends Fig.1: Identification of royal-jelly (RJ) protein fractions and their physical properties. (I) 14% SDS-PAGE analysis of RJ proteins, Lane 1, standard proteins with different molecular weights; Lane 2, 3, 4, 5, crude protein fraction (CPF), PF50 (precipitated protein fraction at 40-50% ammonium sulphate saturation), major RJ protein 2 (MRJP2) isoform X1, and MRJP2, respectively. (II, III) 3D Prediction structures of MRJP2 and

of

MRJP2 X1, respectively. (IV, V) Zeta potential diagram of MRJP2 (-11.1 mV) and

ro

MRJP2 X1 (-14.1 mV) dissolved in milliQ water.

-p

Fig. 2: Flow cytometric analysis to investigate the blocking effect of major royal-jelly protein

re

(MRJP) 2 and its isoform X1 on the cellular Cluster of Differentiation 81 (CD81). (I)

lP

Flow charts of CD81 on the surface of healthy PBMCs before and after treatment with RJ-PFs and the standard drug, Sofosbuvir (SOF). (II) Quantification of the % CD81 an-

na

alyzed by flow cytometry on healthy PBMCs, results are presented as mean ± SE (n=3) and different letters specify the significance at p < 0.05. (III, V) The docked complex of

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CD81 (shown in red-blue) and MRJP2 (shown in yellow) or MRJP2 X1 (shown in green), respectively as obtained by GRAMM-X Protein-Protein Docking Web Server and visualized by Discovery Studio program. The space filling spheres style of the amino acid residues referred to the interacting residues in the docked complex. (IV, VI) The network graph of the docked complex represents the interchain interactions (black square). Res; residues, HI; hydrophobic interaction, V; van der Waals, SB; salt bridge, FEI; favorable electrostatic interaction, UEI; unfavorable electrostatic interaction. Fig. 3: Blocking effect of the major royal-jelly protein (MRJP) 2 and its isoform X1 on the cellular scavenger receptor B type I (SR-B1) compared to Sofosbuvir (SOF). (I) Blocking effect of different concentrations of RJ protein fractions (PFs) to SR-B1. (II)

37

Journal Pre-proof Combination index (CI) plot for the combined fraction of MRJP2 and MRJP2 X1(PF50), "Fa" is the % of SR-B1 blocking. (III) The IC50 values for SR-B1 blocking by RJ-PFs. (IV) Dissociation constant (Kd) and inhibition constant (Ki) values for binding of MRJP2 or its isoform X1 with SR-B1 receptor. (V, VII) The docked complex of SR-B1 (shown in red) and MRJP2 (shown in yellow) or MRJP2 X1 (shown in green), respectively as obtained by GRAMM-X Protein-Protein Docking Web Server and visualized by Discovery Studio program. The space filling spheres style of the

of

amino acid residues referred to the interacting residues in the docked complex. (VI,

ro

VIII) The network graph of the docked complex represents the interchain interactions

-p

(black square). Res; residues, HI; hydrophobic interaction, V; van der Waals, SB; salt

re

bridge, FEI; favorable electrostatic interaction, UEI; unfavorable electrostatic interac-

cance at p < 0.05.

lP

tion. Results are presented as mean ± SE (n=3) and different letters specify the signifi-

na

Fig. 4: Inhibitory effect of major royal-jelly protein (MRJP) 2 and its isoform X1 on the HCV and HBV replication enzymes compared to Sofosbuvir (SOF). (I, II, IV, V) Effect of

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different concentrations of RJ protein fractions (PFs) and SOF on the activities of HCV-NS3/NS4A protease, HCV-NS5B polymerase (POL), HBV DNA-dependent DNA POL (DDDP), and HBV-reverse transcriptase (RT) activity of HBV-POL, respectively. (VI) IC50 values of RJ-PFs and SOF inhibitory actions on the HCV and HBV replication enzymes. (III, VII) Clustered IC50 results of HCV and HBV replication enzymes in hierarchically heat map diagrams, the color distributed from white (low value) to deep orange (high value). (VIII) Combination index (CI) plots for the combined fraction of MRJP2 and MRJP2 X1(PF50), "Fa" is the % inhibition of HCV or HBV replication enzymes. Results are presented as mean ± SE (n=3) and different letters specify the significance at p < 0.05.

38

Journal Pre-proof Fig. 5: Molecular docking of HCV-NS3/NS4A protease and major royal-jelly protein (MRJP) 2 or MRJP2 X1 and interaction characterization. (I, IV) The docked complex of HCVNS3/NS4A protease (shown in red-dark blue-light blue-gray) with MRJP2 (shown in yellow) or MRJP2 X1 (shown in green), respectively as obtained by GRAMM-X Protein-Protein Docking Web Server and visualized by Discovery Studio program, the space filling spheres style of the amino acid residues referred to the interacting residues in the docked complex. (II, V) Network graph of the interchain interactions (black

of

square) between HCV-NS3/NS4A protease chains and MRJP2 or MRJP2 X1, respec-

ro

tively. (III, VI) Interacting amino acid residues (ball and stick atom style) at the binding

-p

site of HCV-NS3/NS4A protease chains (A "red", B "dark blue", C "light blue", D

re

"gray") with MRJP2 or MRJP2 X1, respectively. Res; residues, HI; hydrophobic interaction, V; van der Waals, SB; salt bridge, FEI; favorable electrostatic interaction, UEI;

lP

unfavorable electrostatic interaction.

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Fig. 6: Molecular docking of HCV-NS5B polymerase (POL) and major royal-jelly protein (MRJP) 2 or MRJP2 X1 and interaction characterization. (I, IV) The docked complex of

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HCV-N5B-POL (shown in red-blue) with MRJP2 (shown in yellow) or MRJP2 X1 (shown in green), respectively as obtained by GRAMM-X Protein-Protein Docking Web Server and visualized by Discovery Studio program, the space filling spheres style of the amino acid residues referred to the interacting residues in the docked complex. (II, V) Network graph of the interchain interactions (black square) between HCVNS5B POL and MRJP2 or MRJP2 X1, respectively. (III, VI) Interacting amino acid residues (ball and stick atom style) at the binding site of HCV-NS5B POL chain A (shown in red) with MRJP2 or MRJP2 X1, respectively. Res; residues, HI; hydrophobic interaction, V; van der Waals, SB; salt bridge, FEI; favorable electrostatic interaction, UEI; unfavorable electrostatic interaction.

39

Journal Pre-proof Fig. 7: Molecular docking of HBV-polymerase (POL) and major royal-jelly protein (MRJP) 2 or MRJP2 X1 and interaction characterization. (I, V) The docked complex of HBVPOL domains (brown-pink-red-purple) with MRJP2 (shown in yellow) or MRJP2 X1 (shown in green), respectively as obtained by GRAMM-X Protein-Protein Docking Web Server and visualized by Discovery Studio program, the space filling spheres style of the amino acid residues referred to the interacting residues in the docked complex. (II, VI) Network graph of the interchain interactions (black square) between HBV-POL

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and MRJP2 or MRJP2 X1, respectively. (III, IV) Interacting amino acid residues (ball

ro

and stick atom style) at the binding site of HBV-POL domains (RH "brown", T "pink",

-p

spacer "red", TP "purple") with MRJP2 or MRJP2 X1, respectively. Res; residues, HI;

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hydrophobic interaction, V; van der Waals, SB; salt bridge, FEI; favorable electrostatic interaction, UEI; unfavorable electrostatic interaction, RH; RNase H, T; DNA-

lP

dependent DNA POL/reverse transcriptase domain, TP; terminal protein.

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Fig. 8: Acute toxicity results of protein fraction 50 (PF50) and Sofosbuvir (SOF) in male Albino rats. (I, II) Organ/body weight ratio of rats administered with different doses of PF50

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and SOF, respectively. Results are presented as mean ± SE (n=5) and different letters specify the significance at p < 0.05. (III) Histopathological images of Hematoxylin and Eosin (H&E) stained liver, kidney, lung, and spleen sections from rats administered the lowest (140 mg/kg bw "group I, VI") and the highest (5000 mg/kg bw "group V, X") doses of PF50 and SOF. Arrows refer to abnormal morphological features and their color indicates the following: Yellow; hepatocytes vacuolation and swelling (necrosis), Green; bile duct proliferation, light blue; renal glomerular damage, Dark blue; dilation in the renal tubules, Red; thickening in the alveolar septae, Brown; infiltration of inflammatory cells.

40

Journal Pre-proof Table 1

B (350 mg/kg) 13.00±1.00a

C (700 mg/kg) 12.90±0.10a

D (2500 mg/kg) 13.50±0.50a

E (5000 mg/kg) 12.95±0.05a

RBCs (miL/Cmm)

6.77 ± 0.04a

6.00±0.00a

5.90±1.00a

6.10±0.10a

6.70±1.00a

5.70±1.00a

HCT (%)

38.30± 1.51ac

36.00±1.00ab

35.50±0.00ab

35.00±1.00b

39.90±1.00c

34.20±1.00b

MCV (fL)

54.67±3.18a

55.00±0.03a

54.10±1.00a

49.90±0.00b

53.00±1.00ab

52.00±0.20ab

MCH (Pg)

19.27 ± 0.76a

19.27±0.10a

22.40±0.05b

19.60±0.20ab

20.00±0.05ab

20.00±0.01ab

MCHC (g/dL)

34.43± 2.36a

36.50±0.13a

37.00±0.05a

35.00±0.11a

33.90±1.00a

35.00±1.03a

872.33±53.65a

758.00 ±19.00a

788.00±12.77a

720.00±9.00a

899.00±5.98a

785.00±1.98a

WBCs/ Cmm

3.60±0.21a

2.90±0.00a

2.70±0.20a

2.65±0.05a

2.80±0.10a

2.95±0.05a

Granulocytes (%)

0.53±0.07ab

0.65±0.08b

0.60±0.06ab

0.60±0.00ab

0.90±0.01c

0.40±0.03a

Lymphocytes (%)

94.27±1.29ab

93.35±1.00ab

94.40±5.00ab

96.40±3.98b

92.10±2.76a

92.60±0.77a

Monocytes (%)

3.83±0.23ab

6.00±1.00ab

5.00±0.10ab

3.00±0.80a

7.00±0.13b

7.00±1.00b

Biochemical parameters ALT (IU/mL)

220.44±4.22a

200.03±1.81b

199.22±9.68b

226.85±3.16a

238.64±1.74ac

244.13±2.22c

AST (IU/mL)

503.00±9.24abc

495.50±7.50ab

505.50±3.07abc

483.67±9.08a

517.25±3.45bc

526.00±5.11c

Total proteins (g/dL)

7.86±0.49a

7.93± 0.09a

7.69±0.24a

6.82±0.15a

7.79±0.03a

7.87±0.14a

Albumin (g/dL)

4.56±0.12a

3.90±0.00a

3.95±0.01a

3.10±0.20a

4.264±0.18a

4.25±0.51a

Creatinine (mg/dL)

0.85±0.05a

0.80±0.02a

0.89±0.05a

0.85±0.01a

0.88±0.05a

0.83±0.00a

Urea (mg/dL)

2.48±0.43a

2.39±0.05a

1.98±0.05a

2.00±0.05a

2.07±0.03a

2.07±0.01a

501.55±33.21a

280.04±21.79bc

273.06±9.04b

297.16±4.26bc

313.67±1.39c

285.52±10.63bc

7.65±1.13ab

6.77±0.41a

8.23±0.24b

7.22±0.16ab

7.11±0.46ab

7.14±0.48ab

133.89±27.19a

119.28±0.76b

94.16±1.20c

87.74±1.54d

79.66±2.66e

62.92±2.00f

33.46±3.32a

31.38±0.57b

29.14±0.24c

26.52±0.35d

22.09±0.93e

17.78±0.74f

Sodium (mmol/L) Potassium (mmol/L) TG (mg/dL) Cholesterol (mg/dL)

-p

re

lP

na

Platelet /Cmm

Control

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Hematological Parameters Hb (g/dL)

ro

13.27 ± 0.52a

A (140 mg/kg) 12.80±0.80a

of

Effect of different doses of royal jelly protein fraction 50 (PF50) on some hematological and biochemical parameters in male Albino rats.

Hb, hemoglobin; RBCs, red blood cells; HCT, hematocrit "volume of RBCs in blood"; MCV, mean corpuscular volume "average volume of RBCs"; MCH, mean corpuscular Hb "average mass of Hb/RBCs"; MCHC, mean corpuscular Hb concentration "concentration of Hb in a given volume of RBCs"; WBCs, white blood cells; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglycerides. Results are expressed as mean ± SE (n=5). Different letters are significantly different for the same row at p < 0.05.

41

Journal Pre-proof Table 2

Effect of different doses of Sofosbuvir (SOF) on some hematological and biochemical parameters in male Albino rats. G (350 mg/kg) 14.00±0.40bc

H (700 mg/kg) 12.60±0.00a

I (2500 mg/kg) 14.23±0.37bc

J (5000 mg/kg) 14.63±0.17c

RBCs (miL/Cmm)

6.77 ± 0.04ab

7.18±0.17ac

7.15±0.22ac

6.21±0.00b

7.51±0.36c

7.16±0.16ac

HCT (%)

38.30± 1.51a

37.17±0.47a

43.13±0.27b

38.80±0.00a

44.83±1.37bc

48.90±2.60c

MCV (fL)

54.67±3.18ab

54.67±0.33a

60.33±1.33bc

63.00±0.00bc

60.00±1.00bc

64.00±5.00c

MCH (Pg)

19.27 ± 0.76a

17.70±0.20b

19.60±0.00a

32.40±0.00c

19.00±0.50ab

20.53±0.67a

MCHC (g/dL)

34.43± 2.36a

34.27±0.03a

32.47±0.73ab

20.30±0.00c

28.43±3.17b

30.03±1.33ab

872.33±53.65a

688.00 ±39.00b

681.33±23.67b

793.00±0.00a

424.00±0.00c

665.00±6.00b

WBCs/ Cmm

3.60±0.21a

7.93±0.33b

14.33±0.67c

8.20±0.00b

2.90±0.10a

5.00±0.20d

Granulocytes (%)

0.53±0.07a

1.87±0.77b

1.23±0.23ab

1.10±0.00ab

3.50±0.00c

3.27±0.07c

Lymphocytes (%)

94.27±1.29a

84.13±2.23cd

87.20±2.90bcd

92.80±0.00ab

81.00±3.30d

89.03±0.97abc

Monocytes (%)

3.83±0.23a

13.23±1.47c

14.00±0.00c

6.10±0.00ab

13.23±1.03c

7.70±0.90b

Biochemical parameters ALT (IU/mL)

220.44±4.22a

230.62±1.23a

212.69±4.85a

216.75±6.13a

227.88±11.04a

AST (IU/mL)

503.00±9.24a

539.62±8.70ab

544.38±16.2ab

509.00±21.56a

536.12±11.17ab

580.25±13.13b

Total proteins (g/dL)

7.86±0.49a

9.21±0.38a

9.41±0.33a

7.99±0.04a

7.99±0.30a

7.78±0.16a

Albumin (g/dL)

4.56±0.12a

4.72±0.00a

4.68±0.10a

4.81±0.09a

3.44±0.00b

3.46±0.00b

Creatinine (mg/dL)

0.85±0.05a

0.83±0.05a

0.79±0.08a

0.94±0.11a

0.79±0.09a

0.92±0.15a

Urea (mg/dL)

2.48±0.43a

2.67±0.57a

5.05±0.44b

4.17±0.46b

5.39±0.53b

5.52±0.47b

Sodium (mmol/L) Potassium (mmol/L) TG (mg/dL) Cholesterol (mg/dL)

ro

-p

re

lP

227.96±5.95a

501.55±33.21ab

na

Platelet /Cmm

of

13.27 ± 0.52ab

F (140 mg/kg) 12.70±0.13a

Control

455.04±31.06a

517.37±47.12ab

463.24±32.25a

587.43±8.22bc

649.02±16.53c

7.65±1.13a

6.65±0.48a

7.31±0.52a

5.92±0.65ab

7.45±0.21a

4.57±0.20b

133.89±27.19a

126.94±12.65a

240.83±33.19b

206.94±24.99b

270.00±15.66b

205.00±20.21b

33.46±3.32a

44.79±1.46b

50.39±4.98b

47.75±0.84b

42.19±0.68b

43.18±1.31b

Jo ur

Hematological Parameters Hb (g/dL)

Hb, hemoglobin; RBCs, red blood cells; HCT, hematocrit "volume of RBCs in blood"; MCV, mean corpuscular volume "average volume of RBCs"; MCH, mean corpuscular Hb "average mass of Hb/RBCs"; MCHC, mean corpuscular Hb concentration "concentration of Hb in a given volume of RBCs"; WBCs, white blood cells; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglycerides. Results are expressed as mean ± SE (n=5). Different letters are significantly different for the same row at p < 0.05.

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Journal Pre-proof

Major royal-jelly protein 2 and its isoform X1 are two novel safe inhibitors for hepatitis C and B viral entry and replication Noha H. Habashy a*, Marwa M. Abu-Serie b. Biochemistry Department, Faculty of Science, Alexandria University, Alexandria

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21511, Egypt.

Department of Medical Biotechnology, Genetic Engineering, and Biotechnology Re-

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b

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search Institute, City for Scientific Research and Technology Applications (SRTA-

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City), New Borg EL-Arab 21934, Alexandria, Egypt.

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Highlights

Major royal jelly protein 2 and its isoform X1 are two novel inhibitors of HCV and HBV entry into host cells.

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Major royal jelly protein 2 and its isoform X1 are two novel inhibitors of HCV and HBV replication.

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Major royal jelly protein 2 and its isoform X1 are safe on the vital organs of experimental animals.

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Major royal jelly protein 2 and its isoform X1 are extremely more effective and

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safer than Sofosbuvir drug.

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