Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver

Abstract

In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5610 9 International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1?04 g ml "1 with 73 % of virus below 1?08 g ml "1 . Full-length HCV RNA was detected by Northern blotting and the ratio between positive-and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn 2+ and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7. INTRODUCTIONInfection with hepatitis C virus (HCV) occurs worldwide and afflicts approximately 170 million people (Anonymous, 1997). Chronic infection, which occurs in 80 % of cases, can result in chronic active hepatitis, cirrhosis and hepatocellular carcinoma and, less commonly, extrahepatic autoimmune or immune complex diseases (reviewed by Major et al., 2001). Gene fragments from the virus were initially identified by screening a lambda phage expression library of cDNA from the nucleic acid extracted from infected chimpanzee serum against serum from a non-A non-B hepatitis patient (Choo et al., 1989). Subsequently, the whole virus genome of approximately 9500 nt was rescued, sequenced and shown to exhibit a similar genome organization to the flaviviruses and pestiviruses (Choo et al., 1991). This insight, the in vitro transcription of the genome and extensive studies of viral protein expression in eukaryotic expression systems indicate that the genome consists of a single open reading frame encoding a 3000 aa polyprotein flanked by highly conserved 59 and 39 untranslated regions. The putative structural proteins of the virus, core protein and two membrane glycoproteins, E1 and E2, lie at the amino-terminal end of the polyprotein and are released co-translationally by host cell signal peptidase. The nonstructural proteins of the virus, NS2 to NS5B, are located at the C-terminal end of the polyprotein and are released by virus proteases (reviewed by Major et al., 2001;Bartenschlager & Lohmann, 2000). In between the structural and non-structural...

Association between Hepatitis C Virus and Very-Low-Density Lipoprotein (VLDL)/LDL Analyzed in Iodixanol Density Gradients

“…Posttransplantation, liver function deteriorated and the patient had a retransplantation (17). The transplanted liver (S6b) was removed at retransplantation and found to have a high titer of HCV, 5 ϫ 10 9 IU per gram of liver (39). Serum from patient S6 collected at the time of the retransplantation also contained a high titer of HCV, 6 ϫ 10 8 IU/ml, and was negative by recombinant immunoblot assay and also negative for antibodies against HCV core antigen.…”

“…Patient S6 suffered from common variable immunodeficiency and was infected with the 1a genotype of HCV from contaminated intravenous immunoglobulin with the brand name Gammagard (39). The patient required orthotopic liver transplantation for severe HCV-related cirrhosis and liver failure.…”

High-yield production of a chimeric glycoprotein based on permuted E1 and E2 HCV envelope ectodomains

“…HCV envelope glycoproteins E1 and E2 play an essential role in virus entry into host cells by interacting with cell surface receptors, and inducing fusion between 4 the viral and cellular membranes (Nielsen et al, 2004;Bartosch et al, 2003). E2 is the primary glycoprotein responsible for the interaction with cellular receptors including heparan sulfate, the tetraspanin CD81, the scavenger receptor BI, the tight junction proteins claudin-1 and occludin, the Niemann-Pic C1-like 1 cholesterol absorption receptor and other factors (reviewed in (Ploss and Dubuisson, 2012), while little is known about the exact role of E1 protein.…”

Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein

“…HCV envelope glycoproteins E1 and E2 are components of the viral spike which play an essential role in virus entry into host cells by interacting with cell surface receptors and inducing fusion between the viral and cellular membranes [3,4]. E2 is the primary glycoprotein responsible for receptor interaction, while little is known about the exact role of E1 protein.…”