Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.4 (ribonuclease)
 6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis C virus (HCV) represents one of the major causes of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) around the world. Our knowledge of the life cycle of HCV, however, is limited. Current studies are hampered by the lack of a reproducible, high-level in vitro replication system of HCV. We sought to establish HCV replication in HepG2 cells by gene transfer of in vitro transcribed HCV RNA. In preliminary experiments, diethylaminoethyl-dextran led to more efficient gene transfer than cationic liposomes (lipofectin, lipofectamine, and DOTAP). Therefore, in subsequent experiments, HepG2 cells were transfected with full-length (9.6-kb) and near-full-length (9.4-kb) HCV RNA using diethylaminoethyl-dextran. Transfection with subgenomic HCV RNA and mock transfection were used as controls. Positive- and negative-strand HCV RNA sequences were detected by reverse transcription polymerase chain reaction (KT-PCR) for 60 days in the infectious HCV RNA transfected HepG2 cells. The presence of negative-strand HCV RNA, presumably representing replicative intermediates, was confirmed by ribonuclease protection assay. The intracellular levels of HCV RNA were measured by quantitative competitive RT-PCR from 10 to 50 days after transfection and were stable over this time period at moderately high levels (10(8) to 10(10) genomes per mg of total RNA). Expression of viral core and nonstructural proteins was detected in the cytoplasm of transfected cells by immunostaining. Virus-like particles measuring 50 to 60 nm in diameter were found by electron microscopy in cytoplasmic vesicles and conditioned media of the cells transfected with infectious HCV RNA but not in cells transfected with truncated HCV RNA. Culture supernatants of infectious HCV RNA transfected HepG2 cells were infectious for Daudi cells for three passages tested. The truncated HCV RNA lacking NS5 and 3' untranslated region (3' UTR) of HCV was replication incompetent. This is the first demonstration of HCV particles in HepG2 cells after transfection with infectious HCV RNA. We conclude that we have established a reproducible HCV replication system in HepG2 cells that can be used to study the life cycle of HCV and to test anti-HCV agents.
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PMID:Transfection of HepG2 cells with infectious hepatitis C virus genome. 925 Jan 50

The genus Flavivirus contains more than 70 single-stranded, positive-sense arthropod-borne RNA viruses. Some flaviviruses are particularly medically important to humans and other vertebrates including dengue virus (DENV), West Nile virus, and yellow fever virus. These viruses are transmitted to vertebrates by mosquitoes and other arthropod species. Mosquitoes are also infected by insect-specific flaviviruses (ISFs) that do not appear to be infective to vertebrates. Cell fusing agent virus (CFAV) was the first described ISF, which was discovered in an Aedes aegypti cell culture. We found that while CFAV infection could be significantly reduced by application of RNAi against the NS5 gene, removal of the treatment led to quick restoration of CFAV replication. Interestingly, we found that CFAV infection significantly enhanced replication of DENV, and vice versa, DENV infection significantly enhanced replication of CFAV in mosquito cells. We have shown that CFAV infection leads to increase in the expression of ribonuclease kappa (RNASEK), which is known to promote infection of viruses that rely on endocytosis and pH-dependent entry. Knockdown of RNASEK by dsRNA resulted in reduced DENV replication. Thus, increased expression of RNASEK induced by CFAV is likely to contribute to enhanced DENV replication in CFAV-infected cells.
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PMID:Cell fusing agent virus and dengue virus mutually interact in Aedes aegypti cell lines. 2876 Nov 13