Inhibitory activity of limonoids from Khaya grandifoliola C.DC (Meliaceae) against hepatitis C virus infection in vitro

Document Type : Original Research Article

Authors

1 Department of Biomedical Sciences Faculty of Health Sciences University of Buea South West Region, Buea, Cameroon

2 Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty of Science, University of Yaoundé 1, PO Box 812, Yaoundé, Cameroon

3 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

4 Department of Microbiology, Federal University of Technology, P.M.B 704, Akure, Ondo State, Nigeria

5 Laboratory of Physical Chemistry and Phytochemistry, Department of Organic Chemistry, University of Yaoundé 1, PO Box 812, Yaoundé, Cameroon

Abstract

Objective: A fraction from Khaya grandifoliola has recently been shown to inhibit hepatitis C virus (HCV) infection and three limonoids (17-epi-methyl-6-hydroxylangolensate, 7-deacetoxy-7-oxogedunin and 7-deacetoxy-7R-hydroxygedunin) were purified from this fraction. The present study aimed at assessing the inhibitory effect of these limonoids on HCV using cell-culture derived HCV (HCVcc) system.
Materials and Methods: Cytotoxic effects of the limonoids on Huh7.5 cells were assessed by MTT assay. Huh7.5 cells were transfected with RNA transcripts of the plasmid Jc1/GLuc2a, carrying a Gaussia luciferase reporter gene to rescue the HCVcc particles which were used to infect naïve cells in the presence or absence of the studied limonoids during 72 hr. Infection and replication rates were monitored by luciferase reporter assay and immunofluorescence assay (IFA) while cellular gene expression was analyzed by western blot, respectively.
Results: The limonoids inhibited HCV infection mostly by targeting entry and replication stage. Their inhibitory effect on entry step, comparable to that of anti-CD81 antibody, was related to the blocking of CD81 receptor. In the replication step, the limonoids decreased the expression of NS5B similar to danoprevir. These compounds also significantly decreased but up-regulated the expression of Class-III phosphatidylinositol 4-kinase alpha and 2’,5’-oligoadenylate synthase-3, respectively.
Conclusion: The present findings suggest that limonoids from K. grandifoliola are potential anti-HCV agents and may offer an advantage in the treatment of HCV infection.

Keywords


Averhoff FM, Glass N, Holtzman D. 2012.
Global Burden of Hepatitis C: Considerations
for Healthcare Providers in the United States.
Clin. Infect. Dis. 55, S10–S15.
Bartenschlager R, Cosset FL, Lohmann V, 2010.
Hepatitis C virus replication cycle. J Hepatol,
53: 583–585.
Bartenschlager R, Sparacio S. 2007. Hepatitis C
virus molecular clones and their replication
capacity in vivo and in cell culture. Virus Res,
127: 195–207.
Belouzard S, Cocquerel L, Dubuisson J. 2011.
Hepatitis C virus entry into the hepatocyte.
Open Life Sci, 6.
Bianco A, Reghellin V, Donnici L, Fenu S,
Alvarez R, Baruffa C, Peri F, Pagani M,
Abrignani S, Neddermann P, De Francesco
R. 2012. Metabolism of Phosphatidylinositol
4-Kinase IIIα-Dependent PI4P Is Subverted
by HCV and Is Targeted by a 4-Anilino
Quinazoline with Antiviral Activity. PLoS
Pathog, 8: e1002576.
Borawski J, Troke P, Puyang X, Gibaja V, Zhao
S, Mickanin C, Leighton-Davies J, Wilson
CJ, Myer V, Cornella Taracido I, Baryza J,
Tallarico J, Joberty G, Bantscheff M, Schirle
M, Bouwmeester T, Mathy JE, Lin K,
Anti-HCV effect of Khaya grandifoliola
AJP, Vol. 11, No. 4, Jul-Aug 2021 365
Compton T, Labow M, Wiedmann B, Gaither
LA. 2009. Class III Phosphatidylinositol 4-
Kinase Alpha and Beta Are Novel Host
Factor Regulators of Hepatitis C Virus
Replication. J Virol, 83: 10058–10074.
Cheng YL, Lan KH, Lee WP, Tseng SH, Hung
LR, Lin HC, Lee FY, LeeSD, Lan KH. 2013.
Amiodarone inhibits the entry and assembly
steps of hepatitis C virus life cycle. Clin Sci,
125: 439–448.
Choo Q, Kuo G, Weiner A, Overby L, Bradley
D, Houghton M. 1989. Isolation of a cDNA
clone derived from a blood-borne non-A,
non-B viral hepatitis genome. Science, 244:
359–362.
Galani BRT, Sahuc ME, Sass G, Njayou FN,
Loscher C, Mkounga P, Deloison G, Brodin,
P, Rouillé Y, Tiegs G, Séron K, Moundipa
PF. 2016. Khaya grandifoliola C.DC: a
potential source of active ingredients against
hepatitis C virus in vitro. Arch Virol, 161:
1169–1181.
Gottwein JM, Scheel TKH, Hoegh AM,
Lademann JB, Eugen–Olsen J, Lisby G,
Bukh J. 2007. Robust Hepatitis C Genotype
3a Cell Culture Releasing Adapted
Intergenotypic 3a/2a (S52/JFH1) Viruses.
Gastroenterology, 133: 1614–1626.
Herker E, Harris C, Hernandez C, Carpentier A,
Kaehlcke K, Rosenberg AR, Farese RV, Ott
M. 2010. Efficient hepatitis C virus particle
formation requires diacylglycerol
acyltransferase-1. Nat Med, 16: 1295–1298.
Holzer M, Ziegler S, Albrecht B, Kronenberger
B, Kaul A, Bartenschlager R, Kattner L,
Klein C. Hartmann R. 2008a. Identification
of Terfenadine as an Inhibitor of Human
CD81-Receptor HCV-E2 Interaction:
Synthesis and Structure Optimization.
Molecules, 13: 1081–1110.
Holzer M, Ziegler S, Neugebauer A,
Kronenberger B, Klein CD, Hartmann RW.
2008b. Structural Modifications of
Salicylates: Inhibitors of Human CD81‐
Receptor HCV‐E2 Interaction. Arch Pharm,
(Weinheim) 341: 478–484.
Huang H, Sun F, Owen DM, Li W, Chen Y, Gale
M, Ye J. 2007. Hepatitis C virus production
by human hepatocytes dependent on
assembly and secretion of very low-density
lipoproteins. Proc Natl Acad Sci, 104: 5848–
5853.
Keating GM. 2015. Ledipasvir/Sofosbuvir: A
Review of Its Use in Chronic Hepatitis C.
Drugs, 75: 675–685.
Kouam AF, Yuan F, Njayou FN, He H, Tsayem
RF, Oladejo BO, Song F, Moundipa PF, Gao
GF. 2017. Induction of Mkp-1 and Nuclear
Translocation of Nrf2 by Limonoids from
Khaya grandifoliola C.DC Protect L-02
Hepatocytes against Acetaminophen-Induced
Hepatotoxicity. Front Pharmacol, 8.
Kronenberger B. 2001. Interferon alfa downregulates CD81 in patients with chronic
hepatitis C. Hepatology, 33: 1518–1526.
Kumthip K, Maneekarn N. 2015. The role of
HCV proteins on treatment outcomes. Virol
J, 12.
Lehmann E, El-Tantawy WH, Ocker M,
Bartenschlager R, Lohmann V,
Hashemolhosseini S, Tiegs G, Sass G. 2010.
The heme oxygenase 1 product biliverdin
interferes with hepatitis C virus replication by
increasing antiviral interferon response.
Hepatology, 51: 398–404.
Lin LT, Hsu WC, Lin CC. 2014. Antiviral
Natural Products and Herbal Medicines. J
Tradit Complement Med, 4: 24–35.
Lindenbach BD. 2009. Measuring HCV
Infectivity Produced in Cell Culture and In
Vivo, in: Tang, H. (Ed.), Hepatitis C:
Methods and Protocols. Humana Press,
Totowa, NJ, pp. 329–336.
Lindenbach BD, Evans MJ, Syder AJ, Wolk B,
Tellinghuisen TL, Liu CC, Maruyama T,
Hynes RO, Burton DR, McKeating JA, Rice
CM. 2005. Complete Replication of Hepatitis
C Virus in Cell Culture. Science, 309: 623-
626.
Liu D, Ji J, Ndongwe TP, Michailidis E, Rice
CM, Ralston R, Sarafianos SG. 2015. Fast
Hepatitis C Virus RNA Elimination and
NS5A Redistribution by NS5A Inhibitors
Studied by a Multiplex Assay Approach.
Antimicrob Agents Chemother, 59: 3482–
3492.
Ly KN, Miniño AM, Liu SJ, Roberts H, Hughes
EM, Ward JW, Jiles RB. 2020. Deaths
Associated With Hepatitis C Virus Infection
Among Residents in 50 States and the District
of Columbia, 2016–2017. Clin Infect Dis 71:
1149–1160.
Mohd Hanafiah, K, Groeger J, Flaxman AD,
Wiersma ST. 2013. Global epidemiology of
hepatitis C virus infection: new estimates of
age-specific antibody to HCV
seroprevalence. Hepatol Baltim Md, 57:
1333–1342.
Fondjo Kouam et al.
AJP, Vol. 11, No. 4, Jul-Aug 2021 366
Moradpour D, Penin F, Rice CM. 2007.
Replication of hepatitis C virus. Nat Rev
Microbiol, 5: 453–463.
Njouom R, Texier G, Fontanet A, 2015. O-02:
Prevalence of hepatitis B, hepatitis C and
hepatitis D virus infections in Cameroon:
results from a national population based
survey (The ANRS 12289 project). J Viral
Hepat, 22: 1–2.
Phan T, Beran RKF, Peters C, Lorenz IC,
Lindenbach BD. 2009. Hepatitis C Virus NS2
Protein Contributes to Virus Particle
Assembly via Opposing Epistatic Interactions
with the E1-E2 Glycoprotein and NS3-NS4A
Enzyme Complexes. J Virol, 83: 8379–8395.
Sarrazin C, Zeuzem S. 2010. Resistance to
Direct Antiviral Agents in Patients With
Hepatitis C Virus Infection.
Gastroenterology, 138: 447–462.
Sen GC, Sarkar SN. 2007. The InterferonStimulated Genes: Targets of Direct
Signaling by Interferons, Double-Stranded
RNA, and Viruses, in: Pitha, P.M. (Ed.),
Interferon: The 50th Anniversary. Springer
Berlin Heidelberg, Berlin, Heidelberg, pp.
233–250.
Tellinghuisen TL, Rice CM. 2002. Interaction
between hepatitis C virus proteins and host
cell factors. Curr Opin Microbiol, 5: 419–
427.
Tietcheu Galani BR, Njouom R, Moundipa PF,
2016. Hepatitis C in Cameroon: What is the
progress from 2001 to 2016? J Transl Intern
Med, 4: 162–169.
Wakita T, Pietschmann T, Kato T, Date T,
Miyamoto M, Zhao Z, Murthy K, Habermann
A, Kräusslich HG, Mizokami M,
Bartenschlager R, Liang TJ. 2005. Production
of infectious hepatitis C virus in tissue culture
from a clon