Production of Nonstructural Proteins of Hepatitis C Virus Requires a Putative Viral Protease Encoded by NS3

A human hepatoma cell line constitutively expressing proteins of hepatitis C virus (HCV) was established by transfection with cDNA encoding part of the virus nonstructural (NS) genome region. Proteins consistent with authentic processing at NS3/NS4A, NS4A/NS4B and NS4B/NS5A were identified. Pulse-chase experiments indicated that the cleavage between NS3 and NS4A occurred first and cleavage at other sites followed.Expression of specific surface antigens in response to the presence of HCV proteins was analysed by flow cytometry. A significant increase in CD26 expression was observed in cells expressing the HCV proteins. CD26 plays an important role in cellular signal transduction. Its upregulation in response to the presence of HCV proteins may play a role in viral pathology.

“…Each processed product was also detected by the serum SK (Fig. 2 c) and their sizes were consistent with those reported previously (Grakoui et al, 1993c;Manabe et al, 1994).…”

Section: Establishment Of a Human Liver Cell Line Constitutively Exprsupporting

confidence: 79%

Characterization of an established human hepatoma cell line constitutively expressing non-structural proteins of hepatitis C virus by transfection of viral cDNA

Harada

Kim

Sagawa

et al. 1995

“…Cells were pulse-labelled for 15 min, washed and chased in TC 100 medium for up to 3 h. Samples were harvested at various times and lysed with a solution containing 10 mM-Tri~HC1 (pH 7.8), 150 mM-NaC1, 1 mM-EDTA, 1% NP40, 0"1% SDS and 10 mg/ml of aprotinin. HCV proteins in the cell lysates were immunoprecipitated with a monoclonal antibody (Manabe et al, 1994) against HCV NS5B protein, and the immunocomplexes were collected as described previously (Matsuura et al, 1994). The immunoprecipitates were separated by SDS-PAGE and the radioactivity was measured in a Bio-Image model BAS2000 analyser (Fuji Photo Film).…”

Section: Radiolabelling and Immunoprecipitationmentioning

confidence: 99%

In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses

Suzuki¹,

Sato²,

Chieda³

et al. 1995

By the use of recombinant baculoviruses, the transcleavage of hepatitis C virus (HCV) non-structural polyprotein was studied. The viral serine proteinase encoded by the NS3 gene was expressed efficiently in insect cells infected with a baculovirus recombined with HCV cDNA corresponding to amino acids 1046-1243 and the signal sequence of the rabies virus G protein.Coinfection studies showed the in vivo trans-cleavage activity of the expressed protein by the use of a recombinant producing NS5 as a substrate. We also fou ad that the partially purified NS3 serine proteinase prepared from the recombinant-infected cells could cleave NS5A/5B substrate. Characterization of the proteinase obtained will provide basic knowledge on processing of the HCV polyprotein.

“…In the absence of a satisfactory cell culture system for propagating the virus, studies of its mode of gene expression have depended upon the use of heterologous systems for protein expression, including in vitro translation, transient expression in mammalian cells and production of recombinant vaccinia viruses. By these methods, it has been established that the NS3 region of the polyprotein contains a serine proteinase domain which is responsible for cleavage of the downstream polyprotein in at least four places (Bartenschlager et al, 1993(Bartenschlager et al, , 1994Eckart et al, 1993;Grakoui et al, 1993;Hijikata et al, 1993;Tomei et al, 1993 ;Manabe et al, 1994). By analogy with the yellow fever virus (Chambers et al, 1990) this proteinase is probably essential for the production of infectious virus, and hence represents a possible target for chemotherapeutic intervention.…”

Section: Introductionmentioning

confidence: 99%

Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays

Overton

McMillan

Gillespie

et al. 1995

Recombinant baculoviruses have been constructedwhich express the hepatitis C virus (HCV) NS3 proteinase and its substrates in insect cells. The expressed proteinase has been shown to carry out transcleavage at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions in a cell-based assay. When assayed in a cell-free system using in vitro translated substrates, the proteinase could perform trans-processing of the NS4A/4B and NS5A/5B junctions, but only when coexpressed with NS4A, either as an NS3-4A precursor or by co-infection of cells with NS3-and NS4A-expressing recombinant baculoviruses. Possible reasons for the absolute requirement of the NS3 proteinase for NS4A in vitro are discussed.