Studies Of Ingredient Of Viral Particles And Antiviral Activity Of Interferon Omega By Infectious HCV Cell Cultrue System

Background From 1989 that HCV clone was defined first time,the investigation in the field of HCV was limited by the studying model. Chimpanzee is the only animal model,but because of high expense and other ethical reasons,this huge animal model could not used widely,it was very imperious to construct a celll culture system of HCV.In 1999, Lohmann et al first reported the HCV 1b subgenomic replicon and could uesde for the research of HCV replication,later the HCV pseudopartical came out,it was useful for defining the cellular receptor interacting with HCV membrane proteins.Although the subgenomic replicons and the HCV pseudoparticals were complementary partly,they both could not produce infectious virus,could not imitate the complete whole life cycle of HCV.Till 2005,different four groups reported the HCVcc(HCV celll culture) system that could robustly produce infectious HCV virions,this system benefited from the separation of one viral clone named JFH1(Japanese fulminant hepatitis 1,JFH1),its subgenomic replicon could replicate efficiently without any adaptive mutation.This celll culture system greatly facilitate the investigation of determining the properties and biochemical compositions of HCV virions,studying the whole HCV life cycle and pathogenic mechanism and very importantly,it supplies the platform for antiviral drug selectionObjective To construct the HCV cell culture system and based on HCVcc,exploring the properties and biochemical compositions of HCV virions,and evaluating the antiviral effect of interferon omega.Mathods Transfected the JFH1 HCV full-length RNA (HCV-FL-RNA) produced by reverse genetics into Huh7.5 cells with DMRIE-C liposomal transfection reagent,passaging the transfected cells normally and harvestd the cellular RNA,cellular protein and cell culture supernatant at different time point。Detected HCV RNA and protein by ribozyme protection assay and western blotting,at the same time,infected the na(i|¨)ve Huh7.5 cells with harvested supematant,confirming that this transfection system could produce infectious HCV virions.Based on this system,got a large quantity of supernatant containing infectious HCV particles,after concentration and purification,tht concentrated HCV was subjected to 20 to 60%sucrose gradient sedimentation analysis,a total of 11 fractions with 1 ml each were collected from the bottom to the top of the sucrose gradient,The sucrose in fractions was removed by dilution with 1x phosphate-buffered saline(PBS) at a 1:12 ratio and then ultracentrifugation at 40,000 rpm for 16 h at 4℃in a Beckman SW41 rotor,the resulting HCV pellets were resuspended and aliquoted for subsequent studies.We detected the density and infectivity of each fraction and detected some HCV nonstructure proteins of combined infectious fractions by western blotting.At the same time,we tested the antiviral effect of interferon omega based on HCV 1b replicon and 2a cell culture system.Results Through liposomal transfection reagent we could transfected huge size nucleic acid into susceptible cells.About 2 weeks after transfection passaging,we could detect the obvious HCV RNA and proteins in the transfected cells,and the supernatant collected from the transfected cells could infect the naive Huh7.5 cells confirmed by detecting HCV RNA and proteins in the Huh7.5 cells incubated with the supernatant,the result mentioned above showed that this transfection method could produce infectious HCV virions and we could use this HCV cell culture model to harvest a large quantity of supernatant containing infectious HCV particles.Based on this system,we found that HCV viral particles displayed a large range of densities,varied from 1.08 g/ml to 1.176 g/ml,as revealed by density gradient sedimentation analysis, and the HCV infectivity correlated inversely with the density of HCV particles.We detected apoE protein in some fractions and it showed that apoE amout was correlated with the fraction's infectivity.Interestingly, we also detected some nonstructure protein-NS2,NS3,NS4B,NS5A in the combined infectious fractions,this suggested that except for core,membrane protein 1 and 2,some traditional nonstructure proteins and host proteins such as apoE were also a part of HCV virion.At the same time we tested the antiviral effect of interferon omega based on HCV 1b replicon and 2a cell culture system compared with interferonα-2a,at the same concentration,the HCV RNA and protein levels treated with interferon omega were lower than that treated with interferonα-2a,the difference was statiatic significant(p＜0.05),and based on the HCV RNA analysis,EC50 of interferon omega was lower than interferonα-2a.The infectivity of supernatant from interferon treated groups were decreased compared with control(p＜0.05),especially the group treated with interferon omega.Those difference may be due to the higher level of intracellular STAT1 treated with interferon omega.Conclusions 1.Through liposomal transfection reagent,huge size nucleic acid could be transfected into susceptible cells.2.After HCV-FL-RNA tansfected into Huh7.5 cell lines,the exogenic RNA could replicate automatically and express proteins;the supernatant from transfected cells contains infectious viral particles.3.HCV virions obtained from HCV cell culture system displayed a large range of densities,and the HCV infectivity correlated inversely with the density of HCV particles.4.ApoE was found in some HCV particles and its amount correlated with the infctivity of HCV particles.5,except for structure proteins,some traditional nonstructure proteins including NS2,NS3,NS4B,NS5A coud be detected in HCV virion.6.Interferon omega could suppress the replication of HCV(genotype 1b and 2a),and its antiviral activity was stronger than interferonα-2a,this difference may be due to the stronger stimulation of interferon receptor.