Antiviral Activity of Virocidal Peptide Derived from NS5A Against Two Different HCV Genotypes: An in Vitro Study

LJII #896264, VOL 00, ISS 00

ANTIVIRAL ACTIVITY OF VIROCIDAL PEPTIDE DERIVED FROM NS5A

AGAINST TWO DIFFERENT HCV GENOTYPES: AN IN VITRO STUDY

Reem El-Shenawy, Ashraf Tabll, Noha G. Bader El Din, Yasmine El Abd,

Mohamed Mashaly, Camelia A. Abdel Malak, Reham Dawood, and Mostafa

El-Awady

QUERY SHEET

This page lists questions we have about your paper. The numbers displayed at leftcan be found in the text of the paper for reference. In addition, please review yourpaper as a whole for correctness.

Q1: Au: Please check and confirm all author names, affiliations, and correspon-dence address are correct as set.

Q2: Au: Please clarify what “Dr. Mostafa K. El-Awady, 2007” is. If this is sup-posed to be a citation, it must be added to the reference list in numericalorder and cited in the text in numerical order.

Q3: Au: Please provide the city location for Jena Bioscience GmbH.Q4: Au: In tables 3 and 4, if the author is not paying for color printing, please

add the color name to the “color” column so that it will make sense in blackand white.

Q5: Au: Please check the figure 1 and make sure it is clear. If not, please providea new figure 1 that is 300 dpi or higher resolution.

Q6: Au: Please check the figure 5b and make sure it is clear. If not, pleaseprovide a new figure 1 that is 300 dpi or higher resolution.

Q7: Au: Please check the figure 6b and make sure it is clear. If not, pleaseprovide a new figure 1 that is 300 dpi or higher resolution.

TABLE OF CONTENTS LISTING

The table of contents for the journal will list your paper exactly as it appears below:


AGAINST TWO DIFFERENT HCV GENOTYPES: AN IN VITRO STUDY � ReemEl-Shenawy, Ashraf Tabll, Noha G. Bader El Din, Yasmine El Abd, MohamedMashaly, Camelia A. Abdel Malak, Reham Dawood, and Mostafa El-Awady

Journal of Immunoassay and Immunochemistry, 00:1–18, 2014Copyright © Taylor & Francis Group, LLCISSN: 1532-1819 print/1532-4230 onlineDOI: 10.1080/15321819.2014.896264


AGAINST TWO DIFFERENT HCV GENOTYPES: AN IN VITRO STUDY

Reem El-Shenawy,1 Ashraf Tabll,1 Noha G. Bader El Din,1 Yasmine El Abd,1 Q1Mohamed Mashaly,2 Camelia A. Abdel Malak,2 Reham Dawood,1 and

Mostafa El-Awady1 51Department of Microbial Biotechnology, National Research Center, Giza, Egypt2Department, Faculty of Science, Dameitta University, New Damietta City, Egypt

� This study aimed at assessment of the antiviral activity of an amphipathic α-helical peptidederived from the hepatitis C virus NS5A known as C5A virocidal peptide against different HCVgenotypes. Two sources of HCV virus for in vitro study: HCV genotype 4 sera samples and JFH-1 infectious culture system genotype 2a were used. Several virocidal peptide concentrations weretested to determine the concentration that inhibits HCV propagation in Huh 7.5 cells according tothree different prortocols (pre-infection, coinfection, and post infection). The capacity of the virocidalpeptide to block HCV in Huh7.5 cells infected with different 10 individual serum samples wasevaluated. In the pre-infection protocol, virocidal concentration (20, 50, and 75 μM) showed noviral RNA. In the co-infection protocol, virocidal concentrations (10, 20, 50, 75 μM) showed noviral RNA while in post-infection protocol, 75 μM was the only concentration that blocked the HCVactivity. Results of Huh7.5 cell line transfected with HCV cc J6/JFH and treated with virocidalpeptide revealed that only the higher virocidal concentration (75 μM) showed no amplification. Thepercentage of virocidal blocking in the 10 HCV individual serum samples was 60%. In conclusion,the C5A virocidal peptide has potent antiviral activity against HCV.

10

15

20

Keywords HCV, HCV synthetic peptides, in vitro culture model for HCV

INTRODUCTION

In Egypt more than 93% of HCV infected patients have genotype 4,[1]

which is proved to have poor response to combined pegylated interferon 25alpha-2b and ridavirin therapy. Moreover, only a small percent of EgyptianHCV-infected patients exhibit long term remission with current combined

Address correspondence to Mostafa El Awady, Department of Biotechnology (biomedicalTechnology Group), National Research Center, El-Behooth Street 12622, Dokki, Giza, Egypt. E-mail:[email protected]

Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/ljii.

2 R. El Shenawy et al.

treatment.[2,3] Therefore, development of novel antiviral therapies for HCVinfection is imperative,[4,5] as well as development of cell culture systemsinfected with naturally occurring HCV genotypes which can be used to 30evaluate potential antiviral drugs.

Different studies showed that HCV treatment with antiviral peptide ther-apeutics potentially may reduce the time needed for achievement of stablevirologic responses and be helpful in interferon non responders’ patients.While recent treatment advances of genotype 1 infection using directly act- 35ing antiviral agents (DAAs) are encouraging, there is still a need to developvaccine strategies capable of preventing infection. Moreover, vaccines mayalso be used in future in combination with DAAs enabling interferon-freetreatment regimens.[6,7]

New approaches of HCV target specific antiviral therapy would directly 40block viral replication and prevent continuing infection of liver. Thesepotential therapies include ribozymes,[8] nucleoside analogs,[9] histaminedihydrochloride,[10] inhibitors of viral polymerases, helicases,[11,12]

proteases,[7] antisense oligodeoxynucleotides (AS-ODN),[13,14] andsynthetic peptides.[15−17] 45

Peptide inhibitors are quite attractive candidates for antiviral agents. It isrelatively easy to design a peptide that fits a studied protein, regardless ofthe size and chemical properties of the target site. Several HCV-derived syn-thetic peptides that inhibit HCV infection in the cell culture infection systemwere reported.[6,7,17] One of those inhibitory peptides, a virocidal peptide 50derived from the membrane anchor domain of the HCV nonstructuralprotein NS5A (C5A). Virocidal peptide C5A contains amino acids 3–20 ofthe amphipathic α-helical N-terminal membrane anchor domain of thegenotype 1a HCV NS5A protein. HCV NS5A is an RNA binding protein[18]

and a phosphorylated zinc metalloprotein that is very important for HCV 55replication complex.[19] It was reported by Brass et al. that the N-terminal30 amino acids of NS5A are a membrane anchor and associated with theendoplasmic reticulum (ER).[20]

It has been suggested that C5A recognizes cellular components of thevirus membranes, most likely associated with the membrane lipid. The C5A 60permeabilizes the virus membranes, destabilizes the viral structural integrity,releases viral capsids, and exposes the viral genome to exonucleases fordegradation. The C5A mechanism includes prevention of initiation of infec-tion by destroying the virus and suppressing ongoing infections by blockingthe cell-to-cell spread of the virus.[21,22] 65

The development of HCV replicon technology several years ago[23]

greatly accelerated the pace of antiviral drug discovery, leading to improveddrug discovery further with the establishment of a cell culture model ofHCV infection in 2005[24−26] making it possible to search for inhibitorsof every step in the HCV life cycle and agents that target the virus itself. 70

Antiviral Activity of Peptide Derived from HCV NS5A 3

The current Huh-7-derived HCV virions system uses non naturally occur-ring, cloned HCV genotype 2a strain (JFH-1), cloned HCV genotype 1a(H77-S) containing five adaptive mutations or cloned HCV genotype 1b.The disadvantage of this method is the production of only synthetic RNAexpressed from selected cloned genomic or subgenomic HCV.[27] Earlier 75studies showed that C5A block both HIV and HCV genotype 2 a infections incell culture systems.[15,21] To our knowledge there have not been any studiesperformed on HCV genotype 4, the most common genotype in Egypt.

Here, we evaluate the percent of blocking activity of virocidal peptidederived from HCV NS5A using two different cell culture systems. In the 80first system the cells were directly infected with naturally occurring HCVgenotype 4 from infected patients’ sera. In the second system we used thecell culture system containing HCV cc J6/JFH (HCV genotype 2a replicon).

MATERIALS AND METHODS

Synthesis of the Virocidal Peptide 85

The virocidal peptide was first identified from a peptide library of441 overlapping peptides (18 mers offset by 11 amino acids) covering theentire HCV polyprotein (H77 strain, genotype 1a). The virocidal peptidedesignated as C5A contains residues 3–20 of the amphipathic α helicalpeptide derived from membrane anchor domain of HCV genotype 1a. 90Its structure is (SWLRDIWDWICEVLSDFK) derived from the N termi-nus of HCV NS5A protein.[15] The homology of the C5A peptide withdifferent HCV genotypes was determined. The highly purified virocidalpeptide was synthesized commercially by ANASPEC, Inc. (San Jose, CA,USA), in the amide form, using standard solid-phase synthesis involving 959-flurenylmethoxy carbonyl chemistry and purified using HPLC. Peptideswere reconstituted in 100% Dimethyl Sulfoxide (DMSO) to make (200 xstocks) and stored at -20◦C. Then the peptide stock solution was dilutedto make the final concentration of DMSO reach 0.5% as previouslydescribed.[15] 100

Establishment of Huh7.5 Cell Culture System Infected with HCV

Genotype 4

Huh7.5 cells were a generous gift from Charles Rice (The RockefellerUniversity, USA). The cells were obtained under Material TransferAgreement (MTA #642, Dr. Mostafa K. El-Awady, 2007). Huh7.5 cells were Q2105cultured according to previously described protocol where Huh7.5 cells werecultured till adherent cells became semi-confluent, cells were subcultured


and counted.[17] Then cells were infected with positive HCV serum accord-ing to protocols described by Seipp et al.[28] The total cellular RNA wasextracted using acid guanidinium-phenol-chloroform according to the pro- 110tocol of Chomczynski and Sacchi[29] and Goergen et al.[30] The successfulviral infection in Huh7.5 cells throughout the culture duration was con-firmed at the transcriptional level qualitatively by Reverse TranscriptionPolymerase Chain Reaction (RT-PCR) amplification of HCV RNA and quati-tatively by real-time RT-PCR.[17,31] Moreover, the HCV viral proteins in 115transfected cells were detected using immunostaining assay as previouslydescribed to confirm that direct infection of Huh7.5 cells resulted in viralreplication and de novo synthesis of non structural proteins.[17, 32]

Transfection of Huh7.5 Cell Line with HCV cc J6/JFH

The HCV pJ6/JFH along with its replication deficit derivative pJ6/JFH- 120GND plasmids were obtained from Dr. C. Rice as a generous gift undermaterial transfer agreement (MTA #842 licensed to Dr. Mostafa El-Awady).The HCV cc J6/JFH was prepared according to Tabll et al.[32] Transfectedcells and media were collected after two days and RNA extraction wasperformed from the cell lysate using acid guanidinium-phenol-chloroform 125method according to Chomczynski and Sacchi.[29] HCV RNA detection intransfected Huh7 cells was performed qualitatively by RT-PCR and quatita-tively by real-time RT-PCR. Moreover, the HCV viral proteins in transfectedcells were detected using Indirect Immunofluorescence assay as previouslydescribed.[32] 130

Assessment of Blocking Activity of Virocidal Peptide in Infected

Huh7.5 cell

According to Cheng et al.,[15] cytotoxic activity showed that virocidalpeptide concentration up to 100 μM is non cytotoxic and safe. To deter-mine the virocidal peptide concentration required to inhibit Huh 7.5 cells 135infected with HCV serum, pure virocidal peptide stock solution were dilutedin DMSO (5, 10, 20, 50, 75 μM) and tested for inhibitory activity. Virocidalpeptide was added to Huh7.5 cells according to three different protocols(pre-infection, coinfection, and post-infection). (a) Pre-infection Protocol:different concentrations of the virocidal peptide ranging from (5–75 μM) 140were added to Huh7.5 cells for 4 hr at 37◦C. Then the cells were directlyinfected with pool of 8 HCV genotype 4 infected sera with viral loads (1.5× 106 copies/mL) and incubated in 37◦C incubator over night. (b) Co-infection Protocol: The virocidial peptide was added to cells together withthe virus inoculation. In brief, different concentrations of virocidal peptide 145ranging (5–75 μM) were added to pool of 8 HCV genotype 4 infected sera


with viral load (1.5 × 106 copies/mL). Then the mixture used to inoculatethe cells over night at 37◦C. (c) Post- infection Protocol: the cells wereinfected with pool of 8 HCV genotype 4 infected sera with viral load (1.5× 106 copies/mL) and incubated for 4 hr at 37◦C. Then the different con- 150centration of the virocidal peptide were added to the infected cells rangingfrom (10–75 μM) and incubated overnight at 37◦C. After 24 hr incubationat 37◦C, cells were lysed and the total cellular RNA was extracted from allthe cells and the RNA was subjected to nested RT-PCR analysis.

Sensitivity of the Virocidal Peptide to Block Infection of 155Huh7.5 Cell with Individual HCV Serum Sample

This experiment was designed to evaluate the capacity of the virocidalpeptide to block HCV in Huh7.5 cells infected with different individualserum samples.10 HCV-RNA positive patient sera with different viral loadwere used to test the ability of the virocidal peptide to block HCV in each 160patient, individually. The 10 serum samples were used to infect the culturedHuh7.5 cells in 6-well culture plates and then 75 μM of virocidal peptidewere added. Each sample was used twice with and without virocidal peptide.Cells were harvested, RNA was extracted and HCV RNA was detectedqualitatively by nested RT-PCR and quantitatively by real-time RT-PCR. 165

Assessment of Blocking Activity of Virocidal Peptide in

Transfected Huh7.5 cell

To determine the virocidal peptide concentration required to inhibitHCV replication in transfected Huh7.5 cells, pure virocidal peptide stocksolution were diluted in DMSO and added to cells. In brief, Huh7.5 cells 170tranfected with HCVcc J6/JFH (1.5 × 106 copies/mL) were treated withdifferent virocidial concentration (5, 10, 20, 50, 75 μM) and incubatedovernight at 37◦C. After 24 hr incubation at 37◦C, cells were lysed and thetotal cellular RNA was extracted from all the cells and the RNA was subjectedto nested RT-PCR analysis and real-time-PCR. 175

RESULTS

Synthesis of the Virocidal Peptide

The virocidal peptide designated as C5A contains residues 3–20 of theamphipathic α helical peptide derived from membrane anchor domain ofHCV genotype 1a. Its structure is (SWLRDIWDWICEVLSDFK) derived from 180the N terminus of NS5A protein (H77 strain, HCV genotype 1a). Figure 1


FIGURE 1 Alignment of amino acid sequences of the virocidial peptide sub-genomic regions amongvarious HCV genotypes. Q5

TABLE 1 Homology of synthetic peptides with various HCV genotypes

Homology % HCV genotype

1a_H77NC_004102 100.01b/2k.RU.N687.AY587845 83.33332a-G2AK3.AF169004 72.22223a.DE.HCVCENS1x76918 72.22224a.EG.ED43.Y11604 66.66674a.01-09.DQ418782 72.22225a.GB.EUH1480.Y13184 66.66676a.HK.6a61.DQ480516 83.33337.CA.QC69.EF108306 72.2222

shows the alignment of amino acid sequences of the virocidial peptide sub-genomic regions among various HCV genotypes. Table 1 showed the percentof Homology between virocidal peptide and various HCV genotypes.

Assessment of Blocking Activity of Virocidal Peptide in Infected 185Huh7.5 Cell

The optimum conc. of virocidal peptide to block HCV activity was deter-mined by adding the different virocidal peptide (5, 10, 20, 50, 75 μM) toHuh7.5 cells according to 3 different protocols. In all protocols, uninfectedHuh7.5 cells were used as negative control and Huh7.5 cells inoculated with 190the pool of HCV infected sera were used as positive control. Figure 2 showsRT-PCR products of infected Huh7.5 cells treated with different concentra-tion of virocidal peptide using pre-infection protocol. Results revealed thatvirocidal concentration (20, 50, 75 μM) were the concentrations that showedno viral RNA i.e. the virus was blocked. Figure 3 shows RT-PCR products 195


FIGURE 2 Agarose gel electrophoresis of nested RT-PCR products to show virocidal activity in pre-infection protocol. HCV nested RT-PCR products (at 174 bp band) were visualized on 2% agarose gelsstained with ethidium bromide. Lanes 1–5 represent different conc. of virocidal peptide that were addedbefore infection of the cells. Lane 1 (5 μM) and Lane 2 (10 μM) showed the presence of the viralRNA positive strand, while Lane 3 (20 μM), Lane 4 (50 μM), and Lane 5 (75 μM) showed absence ofviral RNA. M is molecular weight standard DNA marker (50 bp DNA ladder, Jena Bioscience GmbH, Q3Germany). Lane 6 is positive control of pool of infected sera. Lane 7 is negative control.

of infected Huh7.5 cells treated with different concentration of virocidalpeptide using co-infection protocol. Results revealed that virocidal concen-tration (10, 20, 50, 75 μM) were the concentrations that showed no viralRNA, i.e., the virus was blocked. Figure 4 shows RT-PCR products of infectedHuh7.5 cells treated with different concentration of virocidal peptide using 200post-infection protocol. Results revealed that virocidal concentration (75μM) block the virus activity.

Sensitivity of the Virocidal Peptide to Block HCV Replication in

Huh7.5 Cell Infected with Individual Patients Serum

Ten HCV genotype 4a patient sera (positive for both HCV antibodies 205and RNA) were used to test the ability of the virocidal peptide to blockHCV in each patient individually. The 10 serum samples used for individualdirect infection of Huh7.5 cells have different HCV viral load, as shown inTable 2. Figure 5 and Table 3 show RT-PCR products of infected Huh7.5 cellswith individual serum samples and treated with 75 μM of virocidal peptide. 210Results revealed 6 samples were negative by PCR in their respective wells con-taining virocidal peptide, i.e., the virocidal peptide blocks the virus activitywhile in 4 samples the virocidal peptide fails to inhibit HCV activity. Thepercentage of blocking in the 10 samples was 60%.


FIGURE 3 Agarose gel electrophoresis of nested RT-PCR products to show co-infection. HCV nested RT-PCR products (at 174 bp band) were visualized on 2% agarose gels stained with ethidium bromide. Lanes(1–5) infection of cells with pool of patients’ sera previously incubated with different concentrations ofvirocidal peptide. Results showed the presence of the viral RNA positive strand in the cells infected withmixture of patients’ sera and 5 μM virocidal peptide (Lane 1), and showed no viral RNA (i.e., the viruswas blocked or neutralized) in 10 μM virocidal peptide (Lane 2), 20 μM virocidal peptide (Lane 3), 50μM (Lane 4), and 75 μM (Lane 5). M is molecular weight standard DNA marker (50 bp DNA ladder,Jena Bioscience GmbH). Lane 6 is positive control (infected Huh7.5 cells with pool of infected sera).

Assessment of the Blocking Activity of Virocidal Peptide in 215Transfected Huh7.5 Cells

Blocking activity of the virocidal peptide in transfected Huh7.5 cellswas detected using qualitative and quantitative Methods. Figure 6A showsRT-PCR products of transfected Huh7.5 cells treated with different concen-tration of virocidal. The results revealed that higher virocidal concentration 220only (75 μM) block the virus activity. Therefore, RNA copy number in HCVtransfected Huh7.5 cells treated with different virocidal peptide concentra-tion were detected by real-time PCR so any change in viral copy numbers dueto partial or completely inhibition by the virocidal peptide can be detected.Figure 6B and Table 4 show real-time PCR of transfected cells treated with 225virocidal peptide; the results shows gradual decrease when using peptideconcentration from (5–50 μM) without complete block of viral replication


FIGURE 4 Agarose gel electrophoresis of nested RT-PCR products to show post infection. HCV nestedRT-PCR products (at 174 bp band) were visualized on 2% agarose gels stained with ethidium bromide.Lanes (1–5) different conc. of peptide were added after infection of the cells. Lanes 1–4 showed thepresence of the viral RNA positive strand Lane 1 with conc. (5 μM), Lane 2 (10 μM), Lane 3 (20 μM),and Lane 4 (50 μM). While Lane 5 (75 μM) showed absence of viral RNA. Lane 6 is negative control. Mis molecular weight standard DNA marker (50 bp DNA ladder, Jena Bioscience GmbH). Lane 7 is positivecontrol of pool of infected sera.

TABLE 2 Results of samples that infected Huh7 .5 cells, showing its viral load and efficiencyof virocidal inhibition

Patient’s no. Viral load Blocking activity of virocidal peptide

1 1, 200, 000 HCV Blocked (-ve PCR)2 1, 000, 000 HCV Blocked (-ve PCR)3 1, 500, 000 HCV Blocked (-ve PCR)4 950, 000 HCV Blocked (-ve PCR)5 1, 900, 000 HCV not Blocked (+ve PCR)6 2, 670, 000 HCV not Blocked (+ve PCR)7 1, 355, 850 HCV Blocked (-ve PCR)8 650, 700 HCV Blocked (-ve PCR)9 2, 028, 000 HCV not Blocked (+ve PCR)

10 1, 842, 271 HCV not Blocked (+ve PCR)


FIGURE 5 (A) Virocidal ability to inhibit infection of cells treated with individual HCV genotype4 infected patients’ serum samples. HCV nested RT-PCR products (at 174 bp band) were visualized on 2%agarose gels stained with ethidium bromide. Lanes 1–12 cells infected with individual HCV positive serapre incubated with virocidal peptide with (75 μM/mL). Lanes 1, 2, 3, 4, 7, and 8 showed absence of viralRNA. Lanes 5, 6, 9, 10 showed the presence of the viral RNA positive strand. Lane11 is positive controlwhile Lane 12 is negative control. M is molecular weight standard DNA marker (50 bp DNA ladder, JenaBioscience GmbH). (B) Real-time quantitative analysis of viral HCV Huh7.5 cells infected with individualHCV positive patients’ sera pre incubated with 75 μM virocidal peptide. Q6


TABLE 3 Percentage of inhibition as measured by real-time quantitative analysis of viral HCVHuh7.5 cells infected with individual HCV positive patients’ sera pre incubated with 75 μM virocidalpeptide Q4

color name t Calc Conc) L(IU/ml Percentage of inhibition %

1 Patient 1

2 Patient 2

3 Patient 3

4 Patient 4

5 Patient 5 30.31 475000 75%

6 Patient 6 26.49 787650 70.5%

7 Patient 7

8 Patient 8

9 Patient 9 25.76 811200 60%

10 Patient 10 30.70 552681 70%

11 + ve control 31.27 416016

12 −ve control

while higher virocidal concentration only (75 μM) showed no amplifica-tion as the normal uninfected Huh7.5 cells (below the cutoff line), whichindicates complete viral inhibition. 230

DISCUSSION

Peptide inhibitors can inhibit virus attachment, entry, integrity, orreplication. Antiviral agents comprising direct peptide-derived inhibitorsof HCV enzymes such as protease and polymerase have been developed inrecent years.[12] The therapeutic option using C5A as a virocidal peptide 235has received much attention from several investigators around the world.The virocidal peptide (C5A) was reported to prevent the initiation of HCVinfection by destroying the virus and suppresses its replication.[15,22,33]

Here, in the present study we assessed the antiviral activity of virocidalpeptide against different HCV genotypes using two different cell culture 240


FIGURE 6 (A) Virocidal peptide ability to block HCV activity in Huh7.5 cells tranfected with HCVcc.HCV nested RT-PCR products (at 174 bp band) were visualized on 2% agarose gels stained with ethidiumbromide. Lane 1 is replicon positive control and Lane 2 is medium supernatant of transfected cells. Lane3 is negative cell control. Lanes 4–8: different conc. of virocidal peptide (5-75 μM) were added to thetransfected cells with replicon where results shown presence of viral RNA in Lane 4: (5 μM), Lane 5: (10μM), Lane 6: (20 μM), and Lane 7 (50 μM). Only high conc. of virocidal peptide inhibit the virus in cellinfected by replicon where they show absence of HCV RNA in Lane 8 with conc (75 μM). M: is molecularweight standard DNA marker (50 bp DNA ladder, Jena Bioscience GmbH). (B) Real-time quantitativeanalysis of virocidal blocking activity on Huh7.5 cells transfected with HCVcc. Q7

systems. First, we used positive HCV genotype 4 sera sample to directly infectHuh7 cells. Second, we used Huh7 cells transfected with HCV cc J6/JFH(HCV genotype 2a).


TABLE 4 Percentage of inhibition as measured by real-time quantitative analysis of virocidal blockingactivity on Huh7.5 cells transfected with HCVcc

color name ctCalc Conc(IU/mL

Percentage ofinhibition %

1 Huh7.5 cells transfected with HCVcc 25.55 2135472

2 replicon positive control 29.33 147691

3 Media of transfected cells 32.62 907636

4 Huh7.5 cells transfected with HCVccpre incubated with 5 μM virocidalpeptide.

33.61 491158 77%

5 Huh7.5 cells transfected with HCVccpre incubated with 10 μM virocidalpeptide

37.34 395062 81.5%


40.20 177244 91.7 %


40.20 81147 96.2 %

8 Huh7.5 cells transfected with HCVccpre incubated with 75μM virocidalpeptide9 Negative Control

Direct infection of Huh7.5 with infected HCV patient’s sera was cho-sen because it mimics the biology and kinetics of HCV infection when the 245HCV particles invade the hepatoblasts inside the human liver and it pro-duces infectious HCV particles. Also, the patient’s serum contains the wholeviral particle with its quasispecies of different magnitudes of infectivity whilein synthesized HCV RNA particles are usually homogenous. Moreover, theviral replication machinery interacts with host cellular factors present in 250the serum that were found to bind and enhance the HCV replication.[34]

Therefore, in vitro infection of cells with HCV-containing sera can at bestdemonstrate the infectivity of the virus.[32] Results of RT-PCR and real-timePCR confirmed successfully infection of Huh7.5 cells. Also, the results ofimmunostaining assay (data not shown) confirmed that the cultured cells 255were infected as the detection of NS5A usually used as marker of intracellu-lar HCV replication and confirm the de novo HCV non structural proteinsynthesize. These results in agreement with El Awady et al. who establisheddirect HCV infection system and both negative and positive HCV RNAstrands were detectable in cells and supernatant over a period of at least 260three months.[14]

Before designing and synthesis of the virocidal peptide, alignmentof amino acid sequences of C5A virocidal peptide among various HCVgenotypes sequence were done and the results showed that C5A has an


average 69% homology with HCV genotype 4 range from 66–72% with dif- 265ferent genotype 4 strains. On the other hand, the virocidal peptide has72% homology with HCV genotype 2a while it is 100% homology with HCVgenotype 1a.

The infected Huh 7.5 cells were treated with virocidal peptide andconcentration-dependent reduction of HCV RNA (both qualitative and 270quantitative) were demonstrated indicating inhibition of viral translationand replication. Different concentration of virocidal peptides ranging from5–75 μM) were added to infected Huh 7.5 cells using three different proto-cols to assess the blocking and antiviral activity of the C5A virocidal peptide.From the results of the 3 different protocols, it can be concluded that 27575 virocidal peptide completely inhibit HCV activity in infected Huh7.5 cells.Moreover, the results indicated that in the co-infection protocol only lowervirocidal concentration such as 10 μM can inhibit the virus activity andthis can by explained by; when C5A virocidal peptide becomes in contactwith the virus, it permeabilizes liposome membranes, interacts with the viral 280membrane to disrupt its integrity, release viral capsids, and expose the viralgenome to exonucleases for degradation.[35]

Actually, these results in agreement with Cheng et al.,[15] who usedthe previous three protocols regarding the time of virocidal addition oncells transfected with HCV genotype 1a (H77-S) and he found that the 285co-infection protocol gave the best results.[15] To further expand on this pre-liminary conclusion, experiments were designed to assess the capacity andsensitivity of the virocidal peptide to block HCV genotype 4 in Huh7.5 cellsinfected with different individual serum samples. The results showed thatout of the 10 samples, 6 samples were negative by RT-PCR, and real-time 290PCR which indicates the blocking of virus activity by the virocidal peptide.The percentage of virocidal blocking in the 10 serum samples was 60%. Thefailure for blocking of 4 patient serum samples by virocidal peptide couldbe due to high viral load and different quasispecies in the serum sampleswhich may need higher virocidal concentration or longer incubation time. 295A second explanation may be the presence of many proteins and lipids inthe serum that may interact with virocidal peptides and these may lead toa decrease in or an inhibit of the activity of the virocidal peptide, as theantiviral specificity of C5A correlates with its ability to permeabilize liposomemembranes.[15,35] 300

In the second part of this study, the Huh7.5 cell transfected with HCVcc J6/JFH (genotype 2a subgenomic replicon) were used to test the antiviralactivity of the virocidal peptide. Qualitative and quantitative PCR results con-firmed the successful transfection of Huh7.5 with HCV cc J6/JFH (1.5 x106 copies/mL). Also, HCV NS5A protein was detected which confirm the 305transfection of Huh7.5 cells and production de nova NS5A protein insidethe infected cells. Our results are in agreement with Lindenbach et al.,[36]


Moradpour et al.,[37] Tellinghuisen et al.,[38] and Tabll et al.,[32] whoconfirmed that the detected proteins were of the newly synthesized HCVcc.

The blocking activity of the C5A virocidal peptide on transfected 310Huh7.5 cells was evaluated using different concentration of virocidalpeptides. Results revealed that there were gradual decrease when usingpeptide concentration from 5–50 μM but there were no complete blockof viral replication. Only higher virocidal concentration (75 μM) showedno amplification which indicates complete viral inhibition. The discrepancy 315between the virocidal peptide concentrations which were capable for block-ing of virus in HCV genotype 4 infected Huh7.5 cells and HCV genotype2 transfected Huh7.5 cells is not clear. This can be attributed to the dif-ference in HCV genotypes and replication efficiency in the two infectionsystem. As in direct infection the patient’s serum contains the whole viral 320particle with its quasispecies of different magnitudes of infectivity whilein HCV cc J6/JFH RNA particles are usually homogenous with the samedegree of infectivity. Also, the presence of anti HCV Abs in the serumsamples can interfere with viral infectivity and replication into cells,[39,40]

while others reported that antibodies present in the sera from some patients 325can enhance the viral entry and replication.[41,42] The results of this studyshowed that virocidal peptide C5A destabilizes viral structural integrity andhas viral membranolytic activity. It is completely and permanently preventsde novo HCV infection and suppresses ongoing HCV infection in dividingcells. 330

In conclusion, in the absence of an effective vaccine, there is an urgentneed for safe and effective antiviral agents to prevent transmission of HCV.In the present study, an amphipathic α-helical peptide derived from thehepatitis C virus NS5A anchor domain known as C5A virocidal was usedand the results proved that it has potent antiviral activity against HCV. C5A 335exhibits a broad range of antiviral activity against different HCV genotypes,and it inhibits HCV infection by destabilizing HCV virions extracellularlyand within infected cells. The future study will be preclinical experimentsto study the toxicity and effectively of this peptide in small animals (mice orrats). 340

ACKNOWLEDGMENTS

We would like to thank Dr. Charles Rice for providing J6/JFH1 cDNAand Dr. Takaji Wakita for providing JFH1 cDNA.

FUNDING

This work was partially funded through the Science and Technology 345Development Fund (STDF), Egypt-grant no. 894 to Dr. Ashraf Tabll.


REFERENCES

1. Omran, M.H.; Youssef, S.S.; El-Garf, W.T.; Tabll, A.A.; Bader-Eldin, N.G.; Atef, K.; Nabil, W.; El-Awady,M.K. Phylogenetic and Genotyping of Hepatitis C Virus in Egypt. Austral. J. Basic Appl. Sci. 2009, 3,1–8. 350

2. Di Bisceglie, A.M.; Shindo, M; Fong,T.L.; Fried, M.W.; Swain, M.G.; Bergasa, N.V.; Axiotis, C.A.;Waggoner, J.G.; Park, Y.; Hoofnagle, J.H. A Pilot Study of Ribavirin Therapy for Chronic Hepatitis C.Hepatology 1992, 16 , 649–654.

3. Tan, S.L.; Pause, A.; Shi, Y; Sonenberg, N. Hepatitis C Therapeutics: Current Status and EmergingStrategies. Nat. Rev. Drug. Discov. 2002, 1, 867–881. 355

4. Scott, L.J.; Perry, C. M. Interferon-alpha-2b plus Ribavirin: A Review of its Use in the Managementof Chronic Hepatitis C. Drugs 2002, 62, 507–556.

5. Martinand-Mari, C; Lebleu, B; Robbins, L.I. Oligonucleotide-Based Strategies to Inhibit HumanHepatitis C Virus. Oligonucleotides 2003, 13, 539–548.

6. Torresi, J.; Johnson, D.; Wedemeyer, H. Progress in the Development of Preventive and Therapeutic 360Vaccines for Hepatitis C Virus. J. Hepatol . 2011, 54, 1273–1285.

7. Pawlotsky, J.M. Hepatitis C Virus: Standard-of-Care Treatment. Adv. Pharmacol . 2013, 67 , 169–215.8. Macejak, D.G.; Jensen, K.L.; Jamison, S.F.; Domenico, K.; Roberts, E.C.; Chaudhary, N.; Von

Carlowitz, I.; Bellon, L.; Tong, M.J.; Conrad, A.; Pavco, P.A.; Blatt, L.M. Inhibition of Hepatitis C Virus(HCV)-RNA-Dependent Translation and Replication of a Chimeric HCV Poliovirus using Synthetic 365Stabilized Ribozymes. Hepatology 2000, 31, 769–776.

9. Radhakrishnan, S.K.; Layden, T.J.; Gartel, A.L. RNA Interference as a New Strategy against ViralHepatitis. Virology 2004, 323, 173–181.

10. Lalazar, G.; Ilan, Y. Histamine Dihydrochloride: Actions and Efficacy in the Treatment of ChronicHepatitis C Infection. Expert. Rev. Anti. Infect. Ther . 2006, 4, 377–385. 370

11. Cohen, A.J. When HIV and HCV cohabitate. GMHC. Treat. Issues 1999, 13(10), 5–7.12. Stankiewicz-Drogon, A.; Dörner, B.; Erker, T.; Boguszewska-Chachulska, A.M. Synthesis of New

Acridone Derivatives, Inhibitors of NS3 Helicase, which Efficiently and Specifically InhibitSubgenomic HCV replication. J. Med. Chem. 2010, 22, 3117–3126.

13. Kurreck, J. Antisense Technologies. Improvement through Novel Chemical Modifications. Eur. J. 375Biochem. 2003, 270, 1628–1644.

14. El Awady, M.K.; El Abd, Y.S.; Shoeb, H.A.; Tabll, A.A.; Hosny Ael, D.; El Shenawy, R.M.; Atef, K.;Bader El Din, N.G.; Bahgat, M.M. Circulating Viral Core and E1 Antigen Levels as SupplementalMarkers for HCV Chronic Hepatitis. J. Virol . 2006, 3, 67.

15. Cheng, G.; Montero, A.; Gastaminza, P.; Whitten-Bauer, C.; Wieland, S.F.; Isogawa, M.; Fredericksen, 380B.; Selvarajah, S.; Gallay, P.A.; Ghadiri, M.R.; Chisari, F.V. A Virocidal Amphipathic {alpha}-HelicalPeptide that Inhibits Hepatitis C Virus Infection in vitro. Proc. Natl. Acad. Sci. USA 2008, 105,3088–3093.

16. El-Awady, M.K.; Tabll, A .A.; El-Abd, Y. S.; Yousif, H.; Hegab, M.; Reda, M.; El Shenawy, R.; Moustafa,R.I.; Degheidy, N.; El Din, N.G. Conserved Peptides within the E2 Region of Hepatitis C Virus induce 385Humoral and Cellular Responses in Goats. Virol. J . 2009, 6 , 66.

17. El Abd, Y.S.; Tabll, A.A.; El Din, N.G.; Hosny Ael, D.; Moustafa, R.I.; El-Shenawy, R.; Atef, K.; El-Awady,M.K. Neutralizing Activities of Caprine Antibodies towards Conserved Regions of the HCV EnvelopeGlycoprotein E2. Virol. J . 2011, 8, 391.

18. Huang, L.; Hwang, J.; Sharma, S.D.; Hargittai, M.R.; Chen, Y.; Arnold, J.J.; Raney, K.D.; Cameron, 390C.E. Hepatitis C Virus Nonstructural Protein 5A (NS5A) is an RNA Binding Protein. J. Biol. Chem.2005, 280, 36417–36428.

19. Tellinghuisen, T.L.; Marcotrigiano, J.; Rice, C. M. Structure of the Zinc-Binding Domain of anEssential Component of the Hepatitis C Virus Replicase. Nature 2005, 435, 374–379.

20. Brass, V.; Bieck, E.; Montserret, R.; Wölk, B.; Hellings, J.A.; Blum, H.E.; Penin, F.; Moradpour, D. An 395Amino-Terminal Amphipathic Alpha-Helix Mediates Membrane Association of the Hepatitis C VirusNonstructural Protein 5A. J. Biol. Chem. 2002, 277 , 8130–8139.

21. Bobardt, M. D.; Cheng, G.; de Witte, L.; Selvarajah, S.; Chatterji, U.; Sanders-Beer, B.E.; Geijtenbeek,T. B., Chisari, F. V.; Gallay, P.A.; Hepatitis C Virus NS5A Anchor Peptide Disrupts HumanImmunodeficiency Virus. Proc. Natl. Acad. Sci. USA 2008, 105, 5525–5530. 400


22. Zhang, X.; Zhang, N.; Chen, G.; Turpoff, A.; Ren, H.; Takasugi, J.; Morrill, C.; Zhu, J.; Li, C.; Lennox,W.; Paget, S.; Liu, Y.; Almstead, N.; Njoroge, F.G.; Gu, Z.; Komatsu, T.; Clausen, V.; Espiritu, C.;Graci, J.; Colacino, J.; Lahser, F.; Risher, N.; Weetall, M,; Nomeir, A.; Karp, G.M. Discovery of NovelHCV Inhibitors: Synthesis and Biological Activity of 6-(indol-2-yl)pyridine-3-sulfonamides TargetingHepatitis C virus NS4B. Bioorg. Med. Chem. Lett. 2013, 23, 3947–3953. 405

23. Saeed, M.; Scheel, T.; Gottwein, J.; Marukian, S.; Dustin, L.; Bukh, J.; Rice, C. Efficient Replicationof Genotype 3a and 4a Hepatitis C Virus Replicons in Human Hepatoma Cells. Antimicrob. Agents.Chemother . 2012, 56 , 5365–5373.

24. Heller, T.; Saito, S.; Auerbach, J.; Williams, T.; Moreen, T. R.; Jazwinski, A.; Cruz, B.; Jeurkar, N.;Sapp, R.; Luo, G.; Liang, T. J. An in vitro Model of Hepatitis C Virion Production. Proc. Natl. Acad. 410Sci. USA 2005, 102, 2579–2583.

25. Wakita, T.; Pietschmann, T.; Kato, T.; Date, T.; Miyamoto, M.; Zhao, Z.; Murthy, K.; Habermann, A.;Kräusslich, H.G.; Mizokami, M.; Bartenschlager, R.; Liang, T.J. Production of Infectious Hepatitis CVirus in Tissue Culture from a Cloned Viral Genome. Nat. Med. 2005, 11, 791–796.

26. Zhong, J.; Gastaminza, P.; Cheng, G.; Kapadia, S.; Kato, T.; Burton, D.R.; Wieland, S.F.; Uprichard, 415S.L.; Wakita, T.; Chisari, F.V. Robust Hepatitis C virus Infection in vitro. Proc. Natl. Acad. Sci. USA2005, 102, 9294–9299.

27. Buck, M. Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2,3 and 4 in Normal Human Hepatocyte Cultures. PLoS. ONE 2008, 3, 2660.

28. Seipp, S.; Mueller, H.M.; Pfaff, E.; Stremmel, W.; Theilmann, L.; Goeser, T. Establishment of 420Persistent Hepatitis C Virus Infection and Replication in vitro. J .Gen. Virol . 1997, 78, 2467–2476.

29. Chomczynski, P., Sacchi, N. Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem. 1987, 162, 156–159.

30. Goergen, B.; Jakobs, S.; Meyer zum Büschenfelde, K.H.; Gerken, G. Evaluation of Different SerumRNA Extraction Protocols using Quantitative HCV-PCR. J. Hepatol . 1994, 21, 1157. 425

31. El-Awady, M.K.; Ismail, S.M.; El-Sagheer, M.; Sabour, Y.A.; Amr, K.S.; Zaki, E.A. Assay for Hepatitis CVirus in Peripheral Blood Mononuclear Cells Enhances Sensitivity of Diagnosis and Monitoring ofHCV-Associated Hepatitis. Clin .Chim. Acta. 1999, 283, 1–14.

32. Tabll, A.A.; Atef, K.; Bader El Din, N.G.; El Abd, Y.S.; Salem, A.; Sayed, A.A.; Dawood, R. M.;Omran, M.H.; El-Awady, M.K. In vitro Neutralization of HCV by Goat Antibodies against Peptides 430Encompassing Regions Downstream of HVR-1 of E2 glycoprotein. J. Immunoassay Immunochem. 2014,35, 12–25.

33. Lin, Q.; Fang, D.; Hou, X.; Le, Y.; Fang, J.; Wen, F.; Gong, W.; Chen, K.; Wang, J.M.; Su, S.B. HCVPeptide (C5A), an Amphipathic α-Helical Peptide of Hepatitis Virus C, is an Activator of N-formylPeptide Receptor in Human Phagocytes. J. Immunol . 2011, 186 , 2087–2094. 435

34. Spångberg, K.; Wiklund, L.; Schwartz, S. Binding of the La Autoantigen to the Hepatitis C Virus3′ Untranslated Region Protects the RNA from Rapid Degradation in vitro. J. Gen. Virol . 2001, 82,113–120.

35. Stuyver, L.J.; McBrayer, T.R.; Tharnish, P.M.; Clark, J.; Hollecker, L.; Lostia, S.; Nachman, T.; Grier,J.; Bennett, M.A.; Xie, M.Y.; Schinazi, R.F.; Morrey, J.D.; Julander, J.L.; Furman, P.A.; Otto, M.J. 440Inhibition of Hepatitis C Replicon RNA Synthesis by Beta-D-2′-deoxy-2′-fluoro-2′-C-methylcytidine:A Specific Inhibitor of Hepatitis C Virus Replication. Antivir. Chem. Chemother . 2006, 17 , 79–87.

36. Lindenbach, B.D.; Meuleman, P.; Ploss, A.; Vanwolleghem, T.; Syder, A.J.; McKeating, J. A.; Lanford,R.E.; Feinstone, S.M.; Major, M.E.; Leroux-Roels, G.; Rice, C.M. Cell Culture-Grown Hepatitis C Virusis Infectious in vivo and can be Recultured in vitro. Proc. Natl. Acad. Sci. USA 2006, 103, 3805–3809. 445

37. Moradpour, D.; Penin, F.; Rice, C.M. Replication of Hepatitis C Virus. Nat. Rev. Microbiol . 2007, 5,453–463.

38. Tellinghuisen, T.L.; Evans, M.J.; Von Hahn, T.; You, S.; Rice, C.M. Studying Hepatitis C Virus: Makingthe Best of a Bad Virus. J. Virol . 2007, 81, 8853–8867.

39. Gottwein, J.M.; Scheel, T.K.; Jensen, T.B.; Lademann, J.B.; Prentoe, J.C.; Knudsen, M.L.; Hoegh, 450A. M.; Bukh, J. Development and Characterization of Hepatitis C Virus Genotype 1-7 Cell CultureSystems: Role of CD81 and Scavenger Receptor Class B Type I and Effect of Antiviral Drugs.Hepatology 2009, 49 , 364–377.

40. Wakita, T. Hepatitis C Virus Vaccine. Nihon. Rinsho. 2008, 66 , 1961–1964.


41. Lavillette, D.; Morice, Y.; Germanidis, G.; Donot, P.; Soulier, A.; Pagkalos, E.; Sakellariou, G.; Intrator, 455L.; Bartosch, B.; Pawlotsky, J.M.; Cosset, F.L. Human Serum Facilitates Hepatitis C Virus Infection,and Neutralizing Responses Inversely Correlate with Viral Replication Kinetics at the Acute Phase ofHepatitis C Virus Infection. J. Virol . 2005, 79 , 6023–6034.

42. Meyer, K.; Ait-Goughoulte, M.; Keck, Z.Y.; Foung, S.; Ray, R. Antibody-Dependent Enhancement ofHepatitis C Virus Infection. J. Virol . 2008, 82, 2140–2149. 460

Documents

Antiviral Activity of Virocidal Peptide Derived from NS5A Against Two Different HCV Genotypes: An in Vitro Study