Ribosomal Protein S5 Interacts with the Internal Ribosomal Entry Site of Hepatitis C Virus

Abstract

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5 noncoding region called the internal ribosome entry site (IRES).Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of ϳ25 kDa in UV cross-linking experiments. In the present study, we examined the ϳ25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.Translational initiation of most eukaryotic messenger RNAs is mediated by the binding of elongation initiation factor eIF-4 to the modified nucleotide cap on the 5Ј end of the mRNA and the binding of the 40 S ribosomal subunit and other initiation factors (1). However, in hepatitis C virus (HCV) 1 as well as in members of the picornavirus family, the positive-stranded RNA genome is not capped. In these viral genomes, the 5Ј noncoding region (NCR) contains an internal ribosomal entry site (IRES) (2, 3). Trans-acting cellular proteins regulate viral protein synthesis by binding to multiple sites within the IRES (2). For example, a polypyrimidine tract-binding protein (PTB) interacts with several sites in the IRES elements of picornaviruses (e.g. poliovirus (PV), encephalomyocarditis virus, foot-andmouth disease virus, and human rhinovirus) (4 -7). A 52-kDa nuclear factor, La protein, appears to be essential for PV translation initiation, because its addition to La-deficient rabbit reticulocyte lysates stimulates and corrects PV translation (8). In addition to these two proteins, other cellular factors (e.g. the 97-kDa protein and poly(rC)-binding protein-2) are likely to stimulate translation initiation directed by the picornavirus IRES (9, 10).Identification and characterization of IRES-binding proteins are important to the understanding of the mechanisms of internal initiation and, ultimately, to the development of novel therapies for HCV. The binding PTB and La are required for translation by HCV IRES (11, 12). However, Kaminski et al. (13) reported that recombinant PTB did not stimulate HCV IRES function, suggesting that PTB may not be necessary during HCV translation initiation under certain experimental conditions. Despite a considerably lower level of La protein in rabbit reticulocyte lysate, HCV IRES promotes efficient translation activity (14 -16). This clearly differs from the case of translation initiation on the PV genome. Thus, general models proposed for the process of 40 S ribosome entry to IRES elements on the picornavirus genome do not s...

Two internal ribosome entry sites mediate the translation of p53 isoforms

“…Recently, two other proteins, ribosomal protein L26a and nucleolin, have been shown to interact with the p53 5 0 UTR and modulate its translation (Takagi et al, 2005). Interestingly, nucleolin and other ribosomal proteins have been shown to interact with viral IRESs (Fukushi et al, 2001;Izumi et al, 2001). Therefore, the interaction of nucleolin and RPL26a with the p53 mRNA 5 0 UTR might regulate the activities of the p53 IRESs.…”

La Protein Binding at the GCAC Site Near the Initiator AUG Facilitates the Ribosomal Assembly on the Hepatitis C Virus RNA to Influence Internal Ribosome Entry Site-mediated Translation

“…HCV IRES has been shown to be capable of binding directly to purified small ribosomal subunit (40 S) with the help of the ribosomal protein S5, followed by correct positioning on to the initiator AUG (14,18). It is possible that in the absence of any canonical initiation factors, some additional cellular trans-acting factors might also be involved in the formation of functional initiation complex on the HCV IRES RNA.…”

“…Recently, binding of a 25-kDa cellular protein (p25) to HCV IRES has been shown to be important for the efficient translation initiation. p25 was originally suggested to be ribosomal protein S9 but later identified as rpS5 (14,17,18). In fact, HCV IRES has been suggested to have a prokaryotic-like mode of interaction with the 40 S ribosomal subunit, where the 40 S ribosomal subunit is thought to interact with the HCV-IRES through p25 (14).…”

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

“…However, the specific functions of most bacterial ribosomal proteins have yet to be discovered and even less is known about the functions of eukaryotic ribosomal proteins. The functions of eukaryotic ribosomal proteins have recently attracted significant interest not only in connection with their roles in the basic mechanism of protein synthesis, but also in connection with their involvement in some key cellular regulatory processes such as translational control of inflammation (Mazumder et al 2006;Kapasi et al 2007;Ray et al 2007), translation initiation on a subset of IRESs (OdremanMacchioli et al 2000;Fukushi et al 2001;Otto et al 2002;Sarnow et al 2005;Laletina et al 2006;Pfingsten et al 2006;Schuler et al 2006;Nishiyama et al 2007), and the development and progression of diseases such as DiamondBlackfan anemia (Sylvester et al 2004 Gazda andSieff 2006;Morimoto et al 2006).…”