An in vitro model of hepatitis C virus genotype 3a-associated triglycerides accumulation
Introduction
The hepatitis C virus (HCV) is a member of the Flaviviridae family and a major cause of chronic liver disease worldwide [1]. Progression of chronic hepatitis C to cirrhosis and hepatocellular carcinoma is influenced by several cofactors, encompassing male gender, age at infection, excess alcohol drinking [2], obesity [3], coinfection with the hepatitis B virus [4] or the human immunodeficiency virus [5]. Moreover, retrospective studies have shown an association between presence and severity of steatosis and advanced fibrosis [6], [7], [8], [9]. Thus, there is a considerable interest in dissecting the mechanism of steatosis in HCV infection, especially in relationship with other metabolic disturbances associated with HCV, such as insulin resistance [11].
Steatosis is frequent in hepatitis C [12]. Its pathogenesis is due to both viral and host factors. Viral steatosis is mostly reported in patients with genotype 3a, in whom fat accumulation correlates with HCV replication level in serum [6] and liver [13], and disappears after successful antiviral therapy [10], [12], [14], [15], strongly suggesting a direct role of specific viral products in the fat deposition. On the other hand, most steatosis of mild intensity observed in patients infected with genotypes other than 3a seems to recognize a metabolic pathogenesis, its most significant clinical correlate being overweight [6]. The latter kind of steatosis tends to decrease the odds of a virological response to antivirals [10]. Its persistence in patients who respond suggests the lack of a role of HCV in its pathogenesis [10], [14].
HCV has a single stranded, positive polarity RNA encoding for a polyprotein precursor of about 3000 amino acids, which is further cleaved into 10 mature proteins. Among these, the structural protein implicated in the nucleocapsid assembly, referred to as core protein, was shown to induce steatosis in transgenic mice [16] and to be associated with lipid droplets in vitro [17], [18], [19]. The fact that the constructs used in these experimental models were derived from isolates of HCV genotype 1b is in apparent conflict with the clinical observation of the significant association of steatosis with HCV genotype 3a. To address this apparent discrepancy, we established an in vitro model to study the effect of the core protein belonging to several viral genotypes (1b, 2a, 3a, 3h, 4h and 5a) on lipid accumulation.
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Patients
We selected six patients with chronic hepatitis C, infected with HCV genotypes 1 through 5, including our recently characterized genotype 3h from Somalia [20]. Clinical, virological and histological features are shown in Table 1. All patients underwent liver biopsy during diagnostic workup of their HCV infection. At histology, only patient #3, infected with genotype 3a, had severe steatosis (>60% of hepatocytes) that disappeared completely upon successful antiviral therapy, thus proving its
Construction of the HCV core-encoding consensus sequence
Fig. 1 shows the alignment of the six core-encoding consensus sequences considered. The genotypes 3a and 3h differed from each other by as many as 8.9% of residues, as previously reported [19]. The two prolines at positions 138 and 143, required for association of the core protein with lipids [23], are conserved across all genotypes. The YATG motif (residues 164–7), located in the central domain, shared with the plant protein oleosin and required for lipid association [23], is also conserved,
Discussion
Previous data have suggested that the HCV core protein is associated with lipid droplets in vitro [15], [16], [17] and is sufficient to induce liver steatosis in transgenic mice [14]. These observations were made using viral sequences of genotype 1, without providing information on the liver histology of patients from which such isolates were derived. In chronic hepatitis C, steatosis is significantly associated with genotype 3a, although also patients with other genotypes may have
Acknowledgements
This study was supported by Grants 3200-63549.00 and 3200B0-103727 from the Swiss National Science Foundation.
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K.A. and V.P. have contributed equally to this work.
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Current address: Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555-0646, USA.