Tissue culture and animal models for hepatitis C virus Thomas Pietschmann, PhD, Ralf Bartenschlager, PhD  Clinics in Liver Disease  Volume 7, Issue 1, Pages 23-43 (February 2003) DOI: 10.1016/S1089-3261(02)00071-5

Fig. 1 Experimental strategies employed to establish small animal models permissive for hepatitis C virus (HCV) replication. (A) The HCV “Trimera-Mouse”: a beige/nude/X-linked immunodeficient (BNX)-mouse, preconditioned by lethal total body irradiation, is radioprotected by reconstitution with bone marrow cells derived from a severe combined immunodeficient (SCID)-mouse, and subsequently transplanted under the kidney capsule with human liver fragments that had been infected with HCV ex vivo [34]. (B) The HCV “Alb-uPa-chimeric mouse” system. Mice, transgenic for a tandem array of urokinase-type plasminogen activator under the control of the albumin promotor, are crossed with SCID-mice, and both traits are bred to homozygocity. The resulting newborns of this mouse line suffer from liver damage caused by the death of hepatocytes, which allows efficient repopulation with human liver cells after transplantation of primary human hepatocytes. Subsequently, the animals display chimeric mouse-human livers and can be productively infected with HCV [36]. Clinics in Liver Disease 2003 7, 23-43DOI: (10.1016/S1089-3261(02)00071-5)

Fig. 2 Experimental strategy to generate Huh-7 cell lines carrying subgenomic selectable HCV replicons. A schematic representation of the HCV genome is given at the top. The plus-strand RNA molecule carries a single open reading frame (open box) that is flanked at both termini by highly structured nontranslated regions (NTRs) required for replication and translation. The individual mature viral proteins are released by the action of viral and cellular proteases. The structural proteins reside in the N-terminal portion of the polyprotein, whereas the nonstructural proteins required for replication are located in the C-terminal reminder. The organization of selectable subgenomic replicons is depicted below. These RNA molecules are synthesized by in vitro transcription reactions and carry the authentic HCV 5′ and 3′ NTRs, a heterologous internal ribosome entry site (IRES) element directing the expression of the NS3 to NS5B proteins, and a selectable marker protein (neomycin phosphotransferase) expressed via the authentic HCV IRES in the 5′ NTR. Note that the neogene is expressed as a fusion protein with the first 12 amino acids of HCV core to ensure optimal IRES function. Upon transfection, only those cells survive that receive a replicon molecule and sustain replication levels sufficient to confer resistance to the drug G418. These cells proliferate and form a cell colony that can be expanded to a cell line that persistently carries a selectable replicon. Clinics in Liver Disease 2003 7, 23-43DOI: (10.1016/S1089-3261(02)00071-5)

Fig. 3 Identification of determinants influencing replication efficiency of HCV replicons in cell culture. (A) Location of cell culture-adaptive mutations in the HCV polyprotein. Regions that correspond with the NS3 protease (prot) and helicase domains, the NS5B polymerase, and the position of the ISDR within NS5A are indicated by shading. Amino acid substitutions are denoted in the single letter code, and the numbers refer to the positions within the polyprotein of the HCV-Con1 isolate [European Molecular Biology Laboratory (EMBL) data base accession no. AJ238799]. Letters in brackets indicate alternative changes found at the same position. Mutations identified by Blight et al [54] are labeled with an asterisk; the other substitutions have been identified in the authors' laboratory [56,57; R Bartenschlager, unpublished]. As regards S2197P and S2204I, identical mutations were found by both groups. The insertion of a lysine residue at postion 2041 (bold type) was found both in a Con1 and HCV-N replicon-harboring cell line [56,58]. Del2207-2254 represents a deletion of the corresponding residues. (B) The effect of various substitutions on replication in comparison with the wild type sequence (wt) and a replication-incompetent construct with an inactivating mutation in the active center of NS5B (pol). Relative replication efficiency was determined by transient replication assays using subgenomic replicons, carrying the firefly luciferase gene instead of neo. RNAs were transfected into Huh-7 cells, and luciferase activities were determined in cell lysates 4 and 48 h later. The 4h value reflects translation of the input RNA and hence was used to correct for transfection efficiencies [55]. Replication levels are expressed as the percent ratio of luciferase activity obtained 48 h and 4 h after transfection. Luciferase activity after 4 h was set to 100%. Note that certain combinations can be antagonistic (S2197P + R2884G) or cooperative (E1202G + T1280I + S2197P). (C) Influence of host cell condition on replication efficiency. A highly cell culture-adapted reporter replicon was transfected into different passages of the same Huh-7 cell line, and luciferase expression was measured after 4 and 48 h. The replication level is given as in (B). Clinics in Liver Disease 2003 7, 23-43DOI: (10.1016/S1089-3261(02)00071-5)

Fig. 4 Applications of the HCV replicon system. (A) Organization of various replicon constructs. The 4 replicons depicted at the top (I–IV) carry a selectable marker gene (s.m.) like, for instance, neomycin phosphotransferase, and thus can be used to establish cell lines that harbor a persistently replicating HCV RNA. Depending on the construct employed, these cells replicate the entire viral genome (I, selectable full length genome) or subgenomic RNA molecules comprising the viral proteins from NS3-5B (II-IV). In the case of the monocistronic replicon (III), the entire coding region is expressed via the authentic HCV IRES, and the selectable marker protein is fused to the remainder of the polyprotein via a protease cleavage site (shaded box). Measurements of RNA replication in stable replicon-harboring cell lines are facilitated by using replicons in which the selectable marker is fused to a reporter gene via a protease cleavage site (IV). Reverse genetic studies are routinely performed with transient assays based on bicistronic replicons harboring luciferase as reporter gene in the first cistron (V). (B) Schematic representation of HCV life cycle as initiated by RNA transfection and aspects that can be investigated with the replicon system. After transfection of Huh-7 cells with replicon molecules, the polyprotein is expressed and processed in individual products, which form a membrane-associated replicase complex (NS3-5B replicase) that amplifies the viral RNA via a minus-strand intermediate (gray line). In turn, plus-strand RNA progeny serve as the template for the synthesis of new minus-strands, or for polyprotein expression. In case of full-length genomes, the structural proteins are expressed that may assemble into virus particles that egress via the secretory compartment of the cell. Aspects that can be studied with the replicon system are given. Clinics in Liver Disease 2003 7, 23-43DOI: (10.1016/S1089-3261(02)00071-5)