Inhibition of internal ribosomal entry site-directed translation of HCV by recombinant IFN-α correlates with a reduced La protein

Abstract

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5 -nontranslated region (5 NTR)H epatitis C virus (HCV), a positive-strand, enveloped RNA virus, is classified within the genus Hepacivirus of the family Flaviviridae. 1 HCV infects the human liver, leading to the development of chronic hepatitis, cirrhosis, and, in some instances, hepatocellular carcinoma. [2][3][4] The treatment of chronic hepatitis C aims to eliminate viremia, and currently, the antiviral agent interferon alfa (IFN-␣) is commonly used worldwide. [5][6][7] It is known that the antiviral action mechanisms of IFN-␣ chiefly include the transcriptional activation of 2Ј,5Ј-oligoadenylate synthetase (2Ј,5ЈAS) that catalyzes 2Ј,5Ј-oligoadenylate (2Ј,5ЈA) synthesis, 8 thereby activating the endonuclease, RNase L, which degrades viral and cellular RNA (2Ј,5ЈA/RNase L system), and also activating the double-stranded RNA-activated protein kinase (PKR), 9,10 which phosphorylates the initiation factor elF2 and leads to the inhibition of translation. 11,12 However, because IFN is used in various intracellular signal transduction pathways, we speculate that mechanisms of antiviral action by IFN-␣ other than that involving the 2Ј,5ЈA/RNase L system and PKR may exist.It is known that the translation of the HCV-RNA genome is initiated by a highly structured RNA segment, 13,14 the internal ribosome entry site (IRES) that occupies most of the 5Ј nontranslated (5ЈNTR) RNA. [15][16][17] The IRES activity is highly dependent on both the primary sequence of this segment and its ability to form complex secondary and tertiary RNA structures. 18-22 A number of in vitro studies have suggested that several cellular proteins, including both conventional translation initiation factors such as eukaryotic initiation factor 3 (eIF3) 23,24 and noncanonical translation initiation factors such as the nuclear La protein [25][26][27][28][29] or polypyrimidine tract binding protein (PTB), 30-32 may stimulate HCV translation. We previously reported that HCV translation is regulated in a cell-cycle-dependent manner and that cellular proteins that vary in abundance during the cell cycle may be involved in this process, 33 but to date, the mechanism by which HCV translation is regulated in vivo is not well understood.

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

“…Our data clearly show that HCV IRES-driven luciferase expression (but not EMCV IRES-driven luciferase expression) is more resistant than cap-dependent expression to inhibition by PKR. While this work was in progress, similar results were reported by Shimazaki et al (2002) and Rivas-Estilla et al (2002). In the latter case, PKR was actually able to enhance relative HCV IRES activity by three-to fourfold.…”

“…These results suggest that, contrary to the apparent selective resistance of cellular IRES activity to inhibition during apoptosis (Holcik et al 2000a), the HCV IRES may be more sensitive to such inhibition. Similar findings have been reported for the relative responses of HCV IRES-dependent and cap-dependent luciferase activities to the inhibitory effects of increasing cell density, serum starvation, or treatment with interferon in such cell lines (Honda et al 2000a;Shimazaki et al 2002), although conflicting data have also been presented (Koev et al 2002). Thus, although the HCV IRES has PKR-inhibitory properties, these are apparently not sufficient to protect IRES-driven reporter gene expression from inhibition during apoptosis as well as under a variety of other conditions of cell stress.…”

“…To test this, we used an Huh-7 hepatoma cell line stably transfected with a bicistronic plasmid from which Renilla and firefly luciferases are again translated by cap-dependent and HCV IRESdriven mechanisms, respectively (Honda et al 2000a;Shimazaki et al 2002). To induce apoptosis, the Huh-7 cells were treated with TNF␣-related apoptosis-inducing ligand (TRAIL; Srivastava 2001) for 6 h or 22 h. Cell extracts were then prepared and luciferase activities determined.…”

Searching for IRES

“…For example, an early in vitro study had shown that the HCV IRES could function without any noncanonical factors, but recent in vivo studies show that La is required for HCV IRES translation (Shimazaki et al 2002;Costa-Mattioli et al 2004). The RNA binding protein Unr is required for HRV IRES activity, and although all five cold shock domains of Unr are necessary for RNA binding, the binding seems to be a nonspecific sequence interaction (Brown and Jackson 2004).…”

IRES complexity before IFN‐alpha treatment and evolution of the viral load at the early stage of treatment in peripheral blood mononuclear cells from chronic hepatitis C patients

“…However, the antiviral mechanisms by which IFN- induces modifications of specific immune responses and the establishment of non specific immune antiviral state in the infected cells is unknown. It has been suggested that it may act at various stages of the viral cycle and was likely mediated by several biochemical pathways involving various proteins: 2'-5'-oligoadenylate synthethase (2)(3)(4)(5), the Mx proteins and the double-stranded RNA-activated protein kinase RNAdependent (dsRNa-PKR) (Giannelli et al, 1993;He & Katze., 2002;Pawlotsky et al, 1995;Pawlotsky et al, 1996;Samuel., 1998;Samuel., 2001;Shimazaki et al, 2002;Tan & Katze., 2001;Taylor et al, 2005).…”