Resveratrol Affects Hepatitis C Virus Propagation

HCV infection is an escalating global health issue, infection with the hepatitis C virus(HCV) is a major cause of chronic liver disease. Approximately130-170million peopleare chronically infected with HCV, representing2-3%of the world population. The rateof cirrhosis development among people with chronic HCV infection has been estimatedas4-24%after20years, while for HCC it was1–3%after30years. The treatment ofHCV infection has evolved over the years with great progress being made inunderstanding the HCV genome and its molecular virology, culminating in thedevelopment of direct-acting antiviral (DAA) agents targeting HCV viral proteins. Since2011with the development and approval of nine directly acting antiviral drugs, it has ledto a dramatic improvement in therapeutic efficacy with as high as SVR≥90%accompanied by fewer side effects. However, therapy will most likely still remainexpensive as it has been the case for most of the newly developed DAAs, making it farout of reach for most of the needy patients resulting in failure to clear infection in asubstantial proportion of cases. Despite tremendous progress in the treatment of HCV inthe last decade or so, lack of approved vaccine and high cost of the newly developedDAAs necessitate alternative antiviral treatment options.Resveratrol (RVT) is a non-flavonoid polyphenol compound present in many plants andfruits. This phenolic compound was found to be a strong activator of sirtuin, a gene forlongevity and has been implicated as the most important polyphenol responsible for thebeneficial effects of red wine consumption which has been called as the “FrenchParadox”. It has been reported that resveratrol has anti-cancer properties, anti-inflammatory properties, anti-obesity, anti-bacterial properties, and beneficial cardiaceffects. Resveratrol was also shown to improve inflammatory biomarkers in patients withnonalcoholic fatty liver disease. In addition, previous studies also showed that resveratrolinhibits the replication of human cytomegalovirus (HCMV), herpes simplex virus type1(HSV-1), Varicella-zoster virus (VZV) and Influenza A Virus. The polyphenolresveratrol (RVT), a molecule containing two phenyl rings separated by a methylenebridge (Figure8), was the first compound discovered able to mimic CR by stimulating sirtuins[67].Sirtuins1-7(SIRT1-7) belong to the third class of deacetylase enzymes,which are dependent on NAD+for activity. Sirtuins action is linked to gene repression,metabolic control, apoptosis and cell survival, DNA repair, development, inflammation,neuroprotection and healthy aging[52].So far SIRT1has been the most extensivelystudied. AMPK and SIRT1constitute two key targets of RVT. As shown in Figure8, dueto a mild mitochondrial poison, RVT inhibits mitochondrial ATP production, leading to ahigher AMP/ATP ratio and an LKB1-dependent activation of AMPK. Then, AMPKenhances NAD+availability, which would overcome the rate-limitation that this cofactorexerts on SIRT1enzymatic activity.This study aimed to investigate the effect of resveratrol on propagation of HCV lifecycle.In this study we used huh7.5.1cell line to complete our experiment. We began byassessing the cytotoxicity of resveratrol. We performed MTT assay for differentconcentrations of resveratrol. No cytotoxicity was observed for resveratrol at theconcentrations used in our experiments. We also took advantage of Cell-culture–derivedHCV particles (HCVcc), based on a novel monocistronic renilla luciferase reporter virusbased on the intragenotypic genotype2a chimera Jc1, which is designated JcR-2a. JcR-2abased HCVcc are infectious in vitro and therefore, are a widely used model for thecomplete replication cycle of HCV. To evaluate the effects of resveratrol on HCVreplication, we used renilla luciferase reporter viruses (JcR2a). Data from experimentalsettings indicate that resveratrol had no effect RNA replication. Next to assess thepossible influence of resveratrol on virus production, we used the supernatant fromelectroporated and resveratrol-treated cells from the previous experiment to infect na vehuh7.5.1cells and found there was significant decrease in luciferase activity indicatingeffect of resveratrol on HCV attachment/entry and or assembly/release phenotype.Furthermore we also showed that resveratrol does not affect HCV core assembly andrelease by investigating the level of intracellular and extracellular core protein usingELISA. HCV cell entry is a complex multistep process that involves at least4cell type-specific entry factors (CD81, SR-B1, claudin-1, and occludin). HCV entry is essential forinitiation, spread, and maintenance of virus infection and represents an interesting targetfor antiviral therapy. Next to investigate at which step resveratrol inhibits HCV infection,we performed a time of addition experiment. Data showed that pre-treatment of Huh7.5.1 cells with resveratrol did not affect HCV entry indicating resveratrol’s ineffectiveness inaltering the functionality of crucial HCV (co-)receptors. On the other hand co-treatmentresulted in maximum effectiveness indicating the role of resveratrol as anti-HCV byimpairing the ability of the virus itself to enter the target cells. Furthermore to support ourresult we performed plaque assay using uninfected Huh-7.5.1-VISI target cellsharbouring a tagEGFP-NLS-IPS reporter in the presence of resveratrol. Addition of thecompound and HCV at the same time to the cells significantly reduced the number ofplaque formation, suggesting resveratrol likely acts directly against viral particles whichis in agreement with our previous results.