to other flaviviruses. [12][13][14][15] Recently it was demonstrated that Transgenic mice have been produced that express the HCV core can form homodimers and probably multimers 16 hepatitis C virus (HCV) core protein in the liver under similar to the nucleocapsid proteins of retroviruses 17,18 and the transcriptional control of the mouse major urinary HBV. 19,20 In vitro expression of HCV core in different cell protein promoter. These animals express the full length lines has generated conflicting data in terms of protein size, core protein in cytoplasm of their hepatocytes at levels posttranslational modification, and subcellular localizacomparable to those detected in naturally infected pation. 12,15,21,22 In addition, it has recently been reported that tients, without histological or biochemical evidence of HCV core protein transforms NIH 3T3 cells 23 and cooperates liver disease or hepatocellular carcinoma. This contrasts with H-ras in transforming primary rat embryo fibroblasts with recent reports that HCV core protein can transform in vitro. 23 It has also been reported that the transient co-NIH 3T3 cells and cooperates with H-ras to transform expression of HCV core and all of the HBV gene products in primary rat fibroblasts in vitro. Coexpression of HCV HuH-7 cells inhibits HBV gene expression and replication, core protein in double transgenic mice that replicate the suggesting possible intracellular interactions between HCV hepatitis B virus (HBV) does not inhibit hepatocellular core and HBV. 24 HCV E1 and E2 are heavily glycosylated, HBV gene expression or replication, contrary to reports putative envelope proteins. [25][26][27][28][29] They are both believed to be that it inhibits HBV replication in HuH-7 cells after trantransmembrane glycoproteins, with C-terminal hydrophobic sient transfection in vitro. We have also produced anchors. After synthesis and glycosylation, E1 and E2 fold transgenic mice in which a C-terminally truncated and associate primarily through noncovalent interactions (aa384-715) glycosylated HCV E2 protein is expressed in with a slow kinetics as shown in vitro by pulse-chase experithe liver under the transcriptional control of the mouse ments. 30 The HCV glycoproteins are predominantly localized albumin promoter. Despite the high level expression of in the endoplasmic reticulum (ER) and are not translocated HCV E2 protein, no evidence of liver disease was debeyond the cis Golgi. 28,30 Purified E1E2 oligomers have been tected in these animals. These results suggest that the used to successfully immunize chimpanzees against chal-HCV core and E2 proteins are not cytopathic for the lenge with low doses of homologous HCV-1, 31 but it is not hepatocyte in vivo, and they represent an initial step in known if the protection is mediated by neutralizing antibodthe development of a small animal model of HCV immuies or other immune mechanisms. nopathology. (HEPATOLOGY 1997;25:719-727.)
In the absence of an efficient cell culture system and convenient animal models to study HCV pathogenesis, we have Hepatitis C virus (HCV) is a parenterally transmitted hepproduced transgenic mice in which HCV core and HCV E2 atotropic virus that, like hepatitis B virus (HBV), causes are expressed in the liver under the control of the developacute and chronic hepatitis and hepatocellular carcinoma. [1][2][3][4] mentally regulated, liver-specific mouse major urinary pro-HCV is a positive stranded RNA virus whose genome (about tein (MUP) and constitutively active mouse albumin (ALB) 9.6 kb) contains a single uninterrupted open reading frame promoters. The characteristics of these animals form the ba-(ORF) that encodes a polyprotein of 3010-3011 amino sis for the current report. acids. 1,[5][6][7][8] In vitro studies have demonstrated that the polyprotein is processed by cellular and viral proteases to produce
Methods
at least ten different viral structural and nonstructural proteins, whose order is C-E1-E2-E2/p7-NS2-NS3-NS4A-NS4B-Plasmid Construction. Plasmid pGEM3Z-HCV core (M. Houghton, NS5A-NS5B. [9][10][11] unpublished results) contains a BamHI-HindIII fragment that encodes the entire HCV core region (nucleotides 342-914) derived from HCV core protein is a highly basic protein that is well HCV-1 (genotype 1a), 5 extending to amino acid 191, after which a conserved among different HCV isolates. It is thought to reptranslational stop codon has been introduced. To achieve high-level resent the nucleocapsid component of the virion by analogy expression in the liver, we placed the core ORF under the control of the mouse promoter MUP. 32 The BamHI-HindIII fragment was excised from the parental vector, filled in with Klenow polymerase, and subcloned in the unique XbaI cloning site (similarly filled in Abbreviations: HCV, hepatitis C virus; MUP, major urinary protein; HBV, hepatitis B with Klenow polymerase) of plasmid p11AS-SV40-7, downstream of virus; SV40, simian virus 40; ALB, albumin; PCR, polymerase chain reaction; PBS, phosthe mouse MUP promoter, and upstream of the SV40 small T intron phate buffered saline; mRNA, messenger RNA.