Untersuchungen zum Replikationsverhalten des Hepatitis C Virus (HCV) mit manipuliertem 2'-5'Oligoadenylatsynthetase-System
|Other Titles:||Research for replication of Hepatitis C virus in cell culture systems with manipulated 2'-5'oligoadenylate synthetase system||Authors:||Röser, Christina||Supervisor:||Dotzauer, Andreas||1. Expert:||Dotzauer, Andreas||2. Expert:||Stick, Reimer||Abstract:||
The Hepatitis C virus is globally prevalent, with approximately 3 % of the global population chronically infected. According to estimations by the CDC, 20 to 50 % of the chronically infected Hepatitis C patients develop a cirrhosis of the liver. From the cirrhosis in turn, in 25% of all cases liver-cell-carzinomas or hepatic failures emerge. Patients with an acute Hepatis C stand a good chance to cure the disease without treatment, whereas patients with chronic hepatitis are treated with a combined therapy of pegylated interferon and Ribavirin. This treatment achieves long-lasting success-rates up to 60%. Until today, there is no standardised or success-guaranteed therapy.The major problem in Hepatitis C virus research is the lack of a cell culture system. Up to now it did not work, to grow naturally isolates of all Hepatitis C virus subtypes in cell culture. Where effective replication of Hepatitis C virus occured, HCV replicon systems or the highly infectious virus strain JFH-1 were used. All existing HCV replicons contain adaptive mutations in almost all non-structure-proteins. Similarly, JFH-1 is showing noticeable differences to all known HCV subtype 2a sequencies.For these reasons, the intention of this work was to make an initial replication of the Hepatitis C virus in cell culture possible. The primary focus was not to alter the viral RNA. Manipulations should occur instead in the cellular viral defense by overexpression of RNase L inhibitor (RLI). This should disable the RNase L , which works as the effector enzyme for the 2'-5'oligoadenylate synthetase system (oas).For analysation of HCV RNA a specific, sensitive and quantitative HCV detection system was developed. For analysation of cellular genomic expression specific and quantitative real time PCRs were developed to detect the mRNA-quantities of RLI, RNase L, GAPDH as well as real time PCR to quantify 18S rRNA.Infection experiments have been carried out in Huh7 as well as FRhK-4 cells. The RNase L inhibitor was isolated and sequenced from the complete human- and monkey cellular RNA. Evaluation of RLI sequencies showed differences in the nucleic- and amino acid sequence to the RLI reference sequence stored in the NCBI database. Amino acid sequences of human and monkey RNase L inhibitor match completely.Stable overexpression of RLI was neither in Huh7, nor in FRhK-4 cells possible. Whether the RLI-gene was eliminated from the cells or the presence of greater quantities of RLI protein acts lethal upon the cells could not be determined.Transient overexpression of RLI by factors up to 200 were possible. After one week of incubation, increased RLI-mRNA amounts by factors up to 6 could be detected. By multiple transfections, duration of the RLI overexpression could be unrestricted extendet.Hepatitis C virus infection experiments as well as transfection experiments with in vitro transcribed HCV RNA were conducted. By transfection of HCV RNA the critical step of the viral cell entry was bypassed. Neither the HCV infection nor the transfection of in vitro transcibed full length RNA showed any effect on expression of cellular RLI. Overexpression of RLI had in turn no positive effect on HCV RNA replication. Neither in Huh7 nor in FRhK-4 cells the initial replication of Hepatitis C virus with the aid of overexpressed RLI succeeded. Since HCV replication did not occur in RLI-transfected as well as in not transfected cells, no predications about the influence of HCV RNA to the expression of RNase L can be made. Most likely however, the 2Ã¯¿½-5Ã¯¿½oas with the effector enzyme RNase L has no effect on HCV replication in cell culture.
|Keywords:||Hepatitis C virus, cell culture system, Huh7 cells, FRhK-4 cells, RNase L, RNase L inhibitor, 2'-5'oligoadenylate synthetase system||Issue Date:||19-Apr-2007||URN:||urn:nbn:de:gbv:46-diss000106909||Institution:||Universität Bremen||Faculty:||FB2 Biologie/Chemie|
|Appears in Collections:||Dissertationen|
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