Construction Of Chimeric HCV CDNAs And Characterization Of Their RNA Transcripts Infectivity In Vitro

Hepatitis C Virus (HCV), a member of the genus Hepacivirus in family Flaviviridae, is an enveloped positive-strand RNA virus. The 9.6kb genome consists of a single open reading frame (ORF) flanked by 5' untranslated region(UTR) and 3'UTR. HCV is the causative agent of hepatitis C, and most infected individuals are unable to eliminate the virus resulting in a persistent infection up to 75% of total cases, furthermore these patients are at high risk to develop liver fibrosis, liver cirrhosis, or hepatocellular carcinoma. It is estimated that about 170 million people are infected by HCV worldwide, and HCV infection has been viewed as one of the severe global public health problems.No vaccination and effective post-exposure prophylaxis is currently available for hepatitis C, for that the virus is highly heterogeneous and thegenome highly mutable. An additional major obstacle is the lack of a convenient animal model in vivo or an efficient propagation system in vitro, which made it difficult to investigate the virological characteristics of HCV. Up to now there is no breakthrough in the traditional cell culture methods for producing HCV, and a technique which could prepare the transcripts from full-length cDNA corresponding to virus RNA in vitro will greatly enhance the molecular studies of RNA viruses. The possible procedure might includes: ( i ) placing full-length cDNA of virus directly downstream of the RNA polymerase promoter; (ii) transcripting in vitro; (iii) transfecting the transcripts into cells and replicating the virus in cells. The method could not only provide precious information for the genetic expression and replication of natural RNA viruses but also for the induced RNA recombination by mutagenesis, deletion, insertion and complementation perfomance. And it will greatly facilitate the study of HCV replication in cell culture system by the development of genetically engineered RNAs.This research consisted of two parts mainly: ( i )To construct the chimeric full-length HCV cDNAs clone by combining the polyprotein of type la with 5' and 3'UTR of type lb , and then study the infectivity by RNA transcripts in vitro. ( ii )To generate Hepatitis C/Japanese Encephalitis chimeric viruses from cDNA templates in which the envelope proteins gene of the chimeric 1a/1b full-length cDNA clone of HCV were replaced with the counterpart of JEV, and then biological properties of the chimeric RNA transcripts were studied by transfecting into permissive cells in culture. The aim of the sudy was to develope of a chimeric HCV full-length genome which could replicate autonomously in cells.Two plasmids were used to construct the panel of chimeric HCV cDNAs in this study. One is pBRTM 1-3011 (short as p3011 in this report) containing entire ORF of HCV type la strain H77, and internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), but lacking HCV authentic 5'UTR and 3'UTR. 3'UTR is important for HCV infection and RNA replication, so RNA transcribed from p3011 couldn't replicate in cell. The other plasmid pHCVreplbBB7(short as pBB7) is a replicon model of HCV having 5'UTR, non-structural coding sequences(from NS3 to NS5B) and 3'UTR from HCV type lb strain Conl. Because of lacking structural coding sequences, RNA transcribed from pBB7 do not support replication of the intact virus.Standard recombinant DNA technology was used to construct all the plasmids in this study. The DNA fragments containing HCV type lb 5'UTR or 3'UTR were inserted into p3011 respectively, then two recombinant plasimids were generated, one containing ORF from HCV type la, EMCV IRES and 3' UTR from type lb, while the other having polyprotein sequences from HCV type la and 5'UTR and 3'UTR from type lb. The other two near-full-length HCV genomes without 98nt conservative region of 3'UTR were also created to examine the effect of 3'UTR in HCV genome on replication and translation aimed to clear the controversy in previous studies.Firstly the plasmids containing different HCV genome sequences were linearized with Pad restriction enzyme, and RNA transcripts were synthesized in vitro using T7 RNA polymerase, then the full-length and near-full-length HCV RNAs transfection of HepG2 cells were done in the presence of lipofectamine 2000, finally HCV replication was investigated by RT-PCR and Western blot. The results demonstrated that:( i ) Two infectious HCV la/lb chimeric RNAs were obtained. Chimeric full-length RNA containing polyprotein of type la and 5' and 3'UTR of type lb appeared to replicate in HepG2 cells as evidenced by the finding of progeny HCV RNA(negative strands) in cell extracts and by HCV NS3 protein in the transfected cells, and so did the near-full-length HCV RNA. The results unequivocally demonstrated that these HCV RNAs had replicated autonomously. Translation of HCV protein driving by EMCV IRES replacing HCV 5'UTR was unsuccessful in our study, for HCV positive and negative strand in transfected cells couldn't be detected.(ii) It was observed that transfected cells showed significant cytotoxicity, such as the cell density decreasing, morphological changing and viability reducing when the two infectious RNAs replicate and translate in cells. HCV replication fluctuated and diminished almost after 40 days post-transfection.Considering that a favorable cellular environment provided by the host cell to support virus efficient replication and assembly is essential to virus replication apart from the inherited factors of virus itself, while HCV proteins especially envelope proteins may have negative influence on host cell growth and induce apoptosis in host cells, it is presumed that a new HCV cell culture system might be established if the envelope proteins of HCV were altered appropriately. JEV is a Flavivirus genus member of the Flaviviridae family. HCV and JEV share similar even identical features in the genome organization and the protein size. The envelope proteins of the two viruses are glycoproteins, and own the ability to mediate virus attachment and entry into their host cells. In view of the conserved feature of these two viruses, we try to generate chimeric HC/JE viruses from cDNA templates of which the HCVenvelope proteins are substituted with encoded JEV counterpart, and the system is likely to decline the HCV envelope proteins' cytotoxicity, and may produce chimeric viruses with more permissive cell lines and higher replication efficiency. The biological characteristics of the chimera were studied by transfecting HepG2 cells and BHK-21 cells with RNA transcripts, respectively. The results were displayed as followed:(i) HCV/JEV (E) chimeic RNA could transiently replicate in HCV susceptible cell line, HepG2 cells. The transient expression of JEV E protein and HCV NS3 protein were confirmed by ELISA and IFA, respectively. But the chimeic RNA couldn't replicate in JEV susceptible cell line, BHK-21 cells.(ii) The RNA was directly injected into the brain of BALB/c sucking mice and into the liver tissue of BALB/c mice about 25 days old. The mice didn't show any unhealthy symptom, and the specific anti-JEV E protein antibody couldn't be detected in serum, and also JEV E protein and HCV NS3 protein were not found expressed in brain and liver tissue by immuno-histochemistry assay. The chimera may have some defects making replication incompetent in a long term.In summary, the construction of the panel of chimeric HCV cDNA clones and the derived RNA transcripts could provide plentiful and homogeneous viral templates for genetic and functional analyses of HCV replication and exploring the new cell culture system. Although the current study didn't fulfill our final goal, the acquired results in this research could still clue us in on some useful suggestions, that it is absolutely possible to change virus characteristics by altering the viral genome. More work should be done in future study, such as to find more suitable chimera supporting cell lines, and to...