Yellow Fever Virus Replicons as an Expression System for Hepatitis C Virus Structural Proteins

Abstract

Chimeric yellow fever virus (YF) RNAs were constructed in which the YF structural genes were replaced by the hepatitis C virus (HCV) structural genes or fusions between the YF and HCV structural genes. Interestingly, RNA replication required nucleotide complementarity between the 3-located conserved sequence 1 and an RNA sequence located in the 5 end of the YF capsid sequence. The (chimeric-)HCV structural proteins were efficiently expressed and processed, and the native E1/E2 heterodimer was formed. However, no indication for the production of HCV-like particles was obtained. Yellow fever virus (YF) is the type member of the genusFlavivirus in the family Flaviviridae, a group of viruses that also contains the genera Pestivirus and Hepacivirus (for recent reviews, see references 5 and 23). The YF genome is a positivestranded RNA molecule of approximately 11 kb that contains a 5Ј end cap structure. The 3Ј end is nonpolyadenylated but instead contains an extensive secondary RNA structure (17,27,28). The genome encodes for a single polyprotein of over 350 kDa that is co-and posttranslationally processed by host and viral proteases (23). The capsid (C) protein is released from the N-terminal part of this polyprotein by the viral protease NS2B-3. The envelope proteins prM and E are processed by signal peptidase cleavages, while the YF nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) are processed from the remainder of the polyprotein by host proteases and the viral NS2B-3 protease.Hepatitis C virus (HCV) has been classified as a separate genus (Hepacivirus) in the family Flaviviridae. HCV encodes a polyprotein of approximately 3,000 amino acids. Co-and posttranslational cleavage of the polyprotein generates at least 10 polypeptides: the putative structural proteins C, E1, and E2 and several nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). In addition, a small hydrophobic polypeptide (p7) is encoded between the E2 and NS2 genes. The function of this polypeptide is unknown, but studies on the closely related bovine viral diarrhea virus p7 protein indicate that it has a function in virion formation (18). The C protein is the putative nucleocapsid protein, and E1 and E2 are the HCV envelope glycoproteins (15, 25) that form heterodimers stabilized by noncovalent interactions (11). These heterodimers are believed to constitute the native prebudding complex (11) and do not leave the endoplasmic reticulum (ER) due to retention signals present in the C termini of E1 and E2 (8, 9, 14).Studies on HCV have been hampered by the lack of an efficient cell culture system. Recently, a system that allowed the replication of selectable full-length HCV genomes in a hepatoma cell line was established (19,26). Despite the expression of HCV structural proteins, no evidence for the assembly of virions could be obtained.As a tool to study the expression of the HCV structural proteins and possibly HCV assembly, chimeric YF/HCV genomes were constructed in which the structural genes of YF-17D were replaced by the structu...

Hepatitis C Virus Glycoprotein E2 Contains a Membrane-proximal Heptad Repeat Sequence That Is Essential for E1E2 Glycoprotein Heterodimerization and Viral Entry

“…However, additional C-terminal determinants of heterodimerization and glycoprotein function appear to be required because E1 M E2 E heterodimers formed less efficiently than did E1E2 and HIV-1 pseudotyped with the E1 M E2 E chimera was not competent to enter Huh7 cells. Previous studies using a similar chimera, but lacking E2-stem residues 700 -715 adjacent to the TMD, did not result in E1 M E2 E heterodimerization (26). Deletion of residues 700 -715 alters the hydropathy plot of the E1 M E2 E chimera significantly from that of wild type E1E2 (data not shown), which may result in an aberrantly folded heptad repeat region that is unable to mediate heterodimerization.…”

“…Furthermore, heterodimerization is not observed for a chimeric glycoprotein comprising the E1 and E2 ectodomains linked to the yellow fever virus prM and E TMDs, respectively, but lacking residues 700 -715 of the putative E2 stem (26). We therefore asked whether the E2 FIG.…”

E2 of Hepatitis C Virus Inhibits Apoptosis

“…Previously, researchers have investigated the roles of HCV structural proteins using chimeric viruses in which the structural genes of Yellow Fever virus (YFV) or Semliki Forest virus (SFV) were replaced by the structural genes of HCV (52,53). Other studies have used ectopic expression of HCV proteins in cell lines and transgenic mice to investigate the roles of viral proteins (52)(53)(54). The HCV replicon mimicking a part of the natural infection cycle is an alternative model system that could be used in investigation of functions of viral proteins.…”

Incorporation of Tick-Borne Encephalitis Virus Replicons into Virus-Like Particles by a Packaging Cell Line

“…Replicons derived from Kunjin virus, a close relative of West Nile virus, represent the most extensively characterized system, and this has been used successfully both to study flavivirus replication and to develop a promising new tool for heterologous gene expression (19,20,46,47). Replicons have also been derived from West Nile virus, dengue virus, and yellow fever virus (35,36,41).…”