Molecular Dissection of Hepatitis C Virus Expression

Abstract

Objective: The 5′ untranslated region (5′ UTR) of the hepatitis C virus genome is thought to be important for the control of viral gene expression and a likely target for therapeutic interception. A functional role of this viral gene segment was analyzed both in vivo and in vitro. Methods: Transgenic mice carrying a reporter gene that contains the complete 5′ UTR sequence were made. Cellular protein(s) which associate with the 5′ UTR were analyzed by gel shift analysis and a following affinity purification. Results: Transgenic mice revealed protein accumulation only in periportal hepatocytes around the portal triad and not in perivenous hepatocytes around the central vein. Gel shift analysis using mouse liver extracts provides further evidence that trans-acting proteins, which recognize a specific cis-acting element with the 5′ UTR (an apparent stemmed structure formed by two noncontiguous RNA sequences), are present in adult mice but not in young mice. A similar 5′ UTR RNA-protein complex was also detected with human liver extracts. Conclusion: The presence of cellular factor(s) which allow HCV 5′ UTR to express in tissue and differentiation state-specific manner was suggested.

Effect of interferon α and cell cycle progression on translation mediated by the hepatitis C virus 5′ untranslated region: a study using a transgenic mouse model

“…We created this transgenic mouse model, targeting the HCV 5′UTR that controls viral protein synthesis preceding all the events for viral replication [25]. We confirmed that the transgene mRNA covered the entire HCV 5′UTR (Fig.…”

“…Because the albumin promoter driving the expression of the transgene is active throughout the hepatic lobule [27,38] and the expression of albumin in this transgenic animal was not specifically restricted to this area (Fig. 1f), this can be a preference of HCV 5′UTR‐directed translation in these cells, which are in a certain stage of differentiation in the zonation of the hepatic lobule [25].…”

“…Transgenic mice used in this study contain the β ‐galactosidase ( β ‐gal ) fusion gene under the transcriptional control of the mouse albumin enhancer/promoter (Fig. 1a) [25–27]. In brief, the entire HCV 5′UTR (342 nt from genotype 1b) and the adjoining codons for the N‐terminal 16 amino acids of the HCV core protein was ligated in‐frame to the β ‐gal coding sequence, resulting in a 117 kDa protein that has slightly higher molecular weight than original β ‐gal (116 kDa).…”

“… (a) A physical map of the transgenic mouse construct. The mAlb‐5′UTR/lacZ gene, as a 6.7‐kb not I fragment, was microinjected into fertilized mouse eggs to derive transgenic mice [25]. The mouse albumin enhancer/promoter [26–28] sequence is 2.3 kb in length and includes the authentic RNA start‐site [28].…”

“…As viral protein synthesis is the first event upon uncoating of the viral RNA genome, a process which precedes the actual replication of the viral RNA, to interception at this stage is likely to be effective in blocking viral infection. Given the apparent importance of the 5′UTR, we have produced a transgenic mouse model which contains a transgene under the control of the HCV 5′UTR [25]. In this report, we evaluated the effect of IFN α on HCV 5′UTR‐directed translation and examined whether such 5′UTR‐driven translation is regulated by the differentiation state of the hepatocyte.…”