Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system,

Abstract

We cloned and expressed the sequences encoding the structural proteins of the hepatitis C virus in a baculovirus eukaryotic expression system. Four recombinant constructs expressed sufficient hepatitis C virus-specific proteins in insect cell culture to allow analysis of protein cleavage, glycosylation and immunoreactivity. Using immunoblot analysis, we detected a 22-kD protein corresponding to the hepatitis C virus capsid protein cleaved from a larger precursor. Recombinant constructs encoding the presumptive envelope (E1) protein produced products ranging from 30 to 35 kD, whereas constructs encoding the presumptive E2/NS1 protein expressed products ranging in size from 68 to 73 kD. The recombinant envelope proteins were glycosylated, as shown by sensitivity to endoglycosidase F digestion, whereas the capsid was not. We examined the immunoreactivity of these recombinant proteins using sera from 50 patients chronically infected with HCV. Forty-seven of 50 of these sera contained antibodies against the capsid, 14 (28%) also had antibodies against E1 and at least 5 (10%) had antibody against E2/NS1. Forty-seven of 50 sera (94%) were viremic, as determined on hepatitis C virus polymerase chain reaction. The three sera that were hepatitis C virus polymerase chain reaction negative did not have envelope antibodies, whereas all sera that had envelope antibodies were also hepatitis C virus polymerase chain reaction positive. Thus antibodies to baculovirus-expressed hepatitis C virus structural proteins, including E1 and E2/NS1, are found in the presence of viremia.

Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection

“…[10][11][12] In our study, we investiAbbreviations: NS Å not significant (P ú .05); ALT, alanine transgated the antibody response to glycosylated and undeaminase.…”

Hepatitis C Virus E2 Protein Purified from Mammalian Cells Is Frequently Recognized by E2-specific Antibodies in Patient Sera

“…The HCV envelope protein expressed in cells infected with recombinant baculovirus and vaccinia virus was used for detection of envelope-specific antibody in patient sera (17)(18)(19). However, the purification of HCV envelope protein at a high yield was thought to be a difficult task.…”

“…In addition, antibody which neutralizes the binding of E2 to target cell appears to correlate with protection from HCV infection (16). These results suggest that HCV E2 protein is a key viral antigen for a hepatitis C vaccine.The HCV envelope protein expressed in cells infected with recombinant baculovirus and vaccinia virus was used for detection of envelope-specific antibody in patient sera (17)(18)(19). However, the purification of HCV envelope protein at a high yield was thought to be a difficult task.…”

Superinfection Exclusion in Cells Infected with Hepatitis C Virus

“…The recent development of an infectious system that permits the growth of HCV in cell culture (HCVcc) has enabled study of the complete viral life cycle (30,44,51). Both HCVpp and HCVcc entry is mediated by the E1 and E2 envelope glycoproteins, proceeding in a CD81-and pH-dependent manner (23,30,44,50,51).Superinfection exclusion, or homologous interference, is the ability of an established virus infection to interfere with secondary virus infection. Multiple mechanisms that contribute to superinfection exclusion have been demonstrated for various viruses, including interference with receptor-mediated attachment (10,11,41,42) and penetration into cells (39,40,47), as well as downstream replication events (1,22,26).…”