Abstract
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease worldwide. Since several aspects of the infection remain unresolved, there is a pressing need for a convenient animal model that can mimic the clinical disease and aid the evaluation of treatment strategies. Although some success has been achieved in transgenic approaches for development of rodent models of HCV, transgenic expression of the complete HCV polyprotein or an entire set of the viral non-structural (NS) proteins continues to be a serious challenge. Using northern blot and 5′ rapid amplification of cDNA ends (RACE), we unraveled two possible mechanisms that can impede HCV NS transgene expression in the mouse liver. Several truncated transcripts are produced from alternate transcription start sites along the HCV NS sequence within the murine environment, in vivo. Translation of these shorter transcripts is blocked either by the positioning of a contextual stop codon or through a shift in the reading frame. In addition, the complete NS transcript undergoes trans-splicing through 5′ recombination with a non-transgene-derived, spliced leader sequence that appends a potential stop codon upstream of the translation start. These findings thus demonstrate that HCV NS-derived transgenes are subject to aberrant transcriptional initiation and post-transcriptional processing in the nucleus of a mouse host. Strategies to prevent such aberrant transcription start/RNA processing might be key to the development of a successful HCV transgenic mouse model.
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References
Alonzi T, Agrati C, Costabile B et al (2004) Steatosis and intrahepatic lymphocyte recruitment in hepatitis c virus transgenic mice. J Gen Virol 85:1509–1520
Bartenschlager R (2006) Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture. Curr Opin Microbiol 9:416–422
Beard MR, Abell G, Honda M et al (1999) An infectious molecular clone of a Japanese genotype 1b hepatitis c virus. Hepatology 30:316–324
Blight KJ, McKeating JA, Rice CM (2002) Highly permissive cell lines for subgenomic and genomic hepatitis c virus RNA replication. J Virol 76:13001–13014
Duverlie G, Wychowski C (2007) Cell culture systems for the hepatitis C virus. World J Gastroenterol 13:2442–2445
Eng FJ, Walewski JL, Klepper AL et al (2009) Internal initiation stimulates production of p8 minicore, a member of a newly discovered family of hepatitis C virus core protein isoforms. J Virol 83:3104–3114
Frelin L, Brenndorfer ED, Ahlen G et al (2006) The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor alpha mediated liver disease. Gut 55:1475–1483
Frith MC, Ponjavic J, Fredman D et al (2006) Evolutionary turnover of mammalian transcription start sites. Genome Res 16:713–722
Kanegae Y, Lee G, Sato Y et al (1995) Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res 23:3816–3821
Koike K, Moriya K, Matsuura Y (2010) Animal models for hepatitis C and related liver disease. Hepatol Res 40:69–82
Lemon SM, Lerat H, Weinman SA et al (2000) A transgenic mouse model of steatosis and hepatocellular carcinoma associated with chronic hepatitis C virus infection in humans. Trans Am Clin Climatol Assoc 111:146–156; discussion 156–147
Lerat H, Honda M, Beard MR et al (2002) Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology 122(2):352–365
Majumder M, Ghosh AK, Steele R et al (2002) Hepatitis C virus NS5a protein impairs TNF-mediated hepatic apoptosis, but not by an anti-FAS antibody, in transgenic mice. Virology 294:94–105
Mayer MG, Floeter-Winter LM (2005) Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity. Mem Inst Oswaldo Cruz 100:501–513
McGivern DR, Lemon SM (2009) Tumor suppressors, chromosomal instability, and hepatitis C virus-associated liver cancer. Annu Rev Pathol 4:399–415
McPherson CE, Shim EY, Friedman DS et al (1993) An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array. Cell 75:387–398
Pasquinelli C, Shoenberger JM, Chung J et al (1997) Hepatitis C virus core and E2 protein expression in transgenic mice. Hepatology 25:719–727
Postic C, Shiota M, Niswender KD et al (1999) Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274:305–315
Scotto-Lavino E, Du G, Frohman MA (2006a) 3′ end cDNA amplification using classic RACE. Nat Protoc 1:2742–2745
Scotto-Lavino E, Du G, Frohman MA (2006b) 5′ end cDNA amplification using classic RACE. Nat Protoc 1:2555–2562
Smale ST (2001) Core promoters: active contributors to combinatorial gene regulation. Genes Dev 15:2503–2508
Stover NA, Kaye MS, Cavalcanti AR (2006) Spliced leader trans-splicing. Curr Biol 16:R8–R9
Sun JR, Bodola F, Fan XG et al (2001) Hepatitis C virus core and envelope proteins do not suppress the host’s ability to clear a hepatic viral infection. J Virol 75:11992–11998
Sun J, Tumurbaatar B, Jia J et al (2005) Parenchymal expression of CD86/b7.2 contributes to hepatitis C virus-related liver injury. J Virol 79:10730–10739
Tang H, Grise H (2009) Cellular and molecular biology of HCV infection and hepatitis. Clin Sci (Lond) 117:49–65
Vassilaki N, Mavromara P (2009) The HCV ARFP/F/core + 1 protein: production and functional analysis of an unconventional viral product. IUBMB Life 61:739–752
Wakita T, Taya C, Katsume A et al (1998) Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system. J Biol Chem 273:9001–9006
Weber F (2007) Interaction of hepatitis C virus with the type I interferon system. World J Gastroenterol 13:4818–4823
Acknowledgments
This work was supported by National Institutes of Health Grant R01 AI069142 (to J.S.) and John Sealy Memorial Endowment Fund Development Grant (to LLC). M.D. is a James W. McLaughlin Postdoctoral Fellow and a Hans Popper Postdoctoral Fellow of the American Liver Foundation. We thank Dr. James Ellis of NIH for the SAGE analysis, Dr. Stanley Lemon for the FL-N/35 mice, Dr. Junhui Jia for technical support and Dr. Thomas Wood for helpful discussion.
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Jiaren Sun, Teh-sheng Chan contributed equally to this manuscript.
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Desai, M.M., Tumurbataar, B., Zhang, Y. et al. Aberrant transcription and post-transcriptional processing of hepatitis C virus non-structural genes in transgenic mice. Transgenic Res 20, 1273–1284 (2011). https://doi.org/10.1007/s11248-011-9494-x
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DOI: https://doi.org/10.1007/s11248-011-9494-x