Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3′ untranslated region of RNA

Abstract

The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3' untranslated region (UTR). To investigate the effect of the 3'UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA delta, that lacked the RNA region from nt 1286 to 8785. A series of MA delta RNAs that differ in the primary structure of their 3'UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3'X resulted in enhancement of the translation efficiency. Translation of MA delta RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA delta (1041) RNA composed of nt 1 to 1041 but MA delta (8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3'UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA delta RNA.

Hepatitis C virus IRES efficiency is unaffected by the genomic RNA 3′NTR even in the presence of viral structural or non-structural proteins

“…Murakami et al (2001) have observed a suppressor effect of the poly(U/UC) and/or stem-loop III of the 39NTR region on translation efficiency. Only one of the above studies was performed ex vivo (in Huh-7 cells) and demonstrated that IRES activity was stimulated two-to threefold by the 39X region (Ito et al, 1998).…”

“…Indeed, Fang & Moyer (2000) have demonstrated that the presence or absence of the 39NTR sequence did not affect translation efficiency. Moreover, Murakami et al (2001) have observed recently that the complete 39NTR downregulated HCV translation in vitro. This inhibition was removed when the poly(U/UC) or stem-loop III regions of the 39NTR were deleted.…”

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

“…This is the case of flavivirus , certain picornaviruses, such as FMDV (Serrano et al 2006), retroviruses (Ooms et al 2007;Kenyon et al 2008), or Dengue virus (Polacek et al 2009), among others. With respect to HCV, conflicting results regarding the role of the 39 UTR in viral protein synthesis have been reported (Ito et al 1998;Ito and Lai 1999;Fang and Moyer 2000;Michel et al 2001;Murakami et al 2001;Kong and Sarnow 2002;McCaffrey et al 2002;Imbert et al 2003;Bradrick et al 2006;Song et al 2006;Lourenco et al 2008). Today it is commonly accepted that the 39 end exerts an enhancer translational effect through the recruitment of viral and cellular protein factors that stimulate IRES activity (McCaffrey et al 2002;Bradrick et al 2006;Song et al 2006;Lourenco et al 2008).…”

Secondary structure and hybridization accessibility of the hepatitis C virus negative strand RNA 5′‐terminus

“…The 3Ј terminus of the HCV negative strand reportedly plays an analogous role in positive-strand RNA synthesis (25,36,39). The binding of HCV 3Ј termini to various host proteins may exert subtle effects on IRES-mediated translation (16,23,34,55) or protect viral transcripts from degradation by cytoplasmic RNases (15,48,49).The replicative and protein-binding functions of heteropolymeric regions in the HCV 3Ј termini are, in many instances, dependent on the ability of the primary sequence to fold into higher-order RNA structure. In vitro, the X region sequence is capable of adopting a three-stem-loop structure, with the 3Ј-terminal 46 nt forming a thermodynamically stable stem-loop, SL I (6).…”

“…The 3Ј terminus of the HCV negative strand reportedly plays an analogous role in positive-strand RNA synthesis (25,36,39). The binding of HCV 3Ј termini to various host proteins may exert subtle effects on IRES-mediated translation (16,23,34,55) or protect viral transcripts from degradation by cytoplasmic RNases (15,48,49).…”