Hepatitis C Virus Core Protein Acts as a trans-Modulating Factor on Internal Translation Initiation of the Viral RNA

Abstract

Translation initiation of hepatitis C virus (HCV) RNA occurs through an internal ribosome entry site (IRES) located at its 5 end. As a positive-stranded virus, HCV uses the genomic RNA template for translation and replication, but the transition between these two processes remains poorly understood. HCV core protein (HCV-C) has been proposed as a good candidate to modulate such a regulation. However, current data are still the subject of controversy in attributing any potential role in HCV translation to the HCV core protein. Here we demonstrate that HCV-C displays binding activities toward both HCV IRES and the 40 S ribosomal subunit by using centrifugation on sucrose gradients. To gain further insight into these interactions, we investigated the effect of exogenous addition of purified HCV-C on HCV IRES activity by using an in vitro reporter assay. We found that HCV IRES-mediated translation was specifically modulated by HCV-C provided in trans, in a dose-dependent manner, with up to a 5-fold stimulation of the IRES efficiency upon addition of low amounts of HCV-C, followed by a decrease at high doses. Interestingly, mutations within some domains of the IRES as well as the presence of an upstream reporter gene both lead to changes in the expected effects, consistent with the high dependence of HCV IRES function on its overall structure. Collectively, these results indicate that the HCV core protein is involved in a tight modulation of HCV translation initiation, depending on its concentration, and they suggest an important biological role of this protein in viral gene expression. Hepatitis C virus (HCV)1 is the primary causative agent of non-A and non-B hepatitis, frequently associated with high rates of progressive liver disease, leading to cirrhosis and hepatocellular carcinoma (1). Since its identification in 1989 (2), appreciation of the significant worldwide impact of chronic HCV infection has grown as ϳ170 million people, i.e. 3% of the world's population, are infected with HCV. HCV is an enveloped positive-stranded RNA virus whose genome of about 9600 nucleotides (nt) encodes a polyprotein that is processed by both host and viral proteases to yield individual structural and nonstructural HCV proteins (3).HCV, along with pestiviruses and GB virus B (GBV-B), are members of the Flaviviridae family that utilize a cap-independent mechanism to initiate translation of their viral RNA that starts at the AUG codon (position 341 for HCV), following ribosome recognition of the upstream internal ribosome entry site (IRES) (4). HCV IRES is a cis-acting element, highly folded into a complex secondary and tertiary structure consisting of several stem-loop domains, which has been shown to be crucial for its functionality (reviewed in Ref. 5). Of the four domains delineated within the HCV 5Ј-untranslated region (5Ј-UTR), only the first seems not to be part of the IRES. The minimal sequence necessary for IRES activity extends approximately from nt 42 to a short stretch (12-30 nt) downstream of the initiation codon (6). H...

Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

“…A capped RNA encoding Renilla luciferase (RLuc) was used as an internal control to normalize transfection efficiency. The validity of similar systems in addressing IRESdependent translation mechanisms has been shown previously (Boni et al, 2005;Shimoike et al, 1999). Total RNA was extracted from CSFV strain Shimen.…”

The current understanding of Ded1p/DDX3 homologs from yeast to human

“…DDX3 is known to interact with the hepatitis C virus (HCV) core protein, [40][41][42] which can differentially modulate the internal ribosome entry site (IRES)-mediated translation of the HCV RNA. 43,44 A recent report shows that overexpression of DDX3, despite its suppressing effect on cap-dependent translation, can activate the initiation of HCV …”

Modulation of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3 by Hepatitis C Virus Core Protein

“…This result further supported that MAPKAPK3 modulated the HCV protein level but not the intracellular HCV RNA level. Since HCV IRES-mediated translation was specifically modulated by HCV core protein in previous studies (23,24) and knockdown of MAPKAPK3 significantly decreased HCV IRES activity in the presence of core protein in the current study, these data indicated that modulation of HCV IRES-mediated translation by core protein occurred through MAPKAPK3 protein. Moreover, overexpression of mutant core containing the necessary region to bind to MAPKAPK3 competed away the protein interaction between wild-type core and MAPKAPK3, and hence HCV IRES activity was impaired.…”

“…These data indicate that MAPKAPK3 is involved in HCV IRES-mediated translation. It has been previously reported that HCV core stimulated HCV IRES activity (23,24). Indeed, overexpression of core protein increased HCV IRES activity 1.5-fold compared to vector control, and core-mediated IRES activity was further elevated by MAPKAPK3 in a dose-dependent manner (Fig.…”