A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site-Dependent Translation through an Interaction Downstream of the Hepatitis C Virus Polyprotein Initiation Codon

Abstract

Translational initiation of hepatitis C virus (HCV) mRNA occurs by internal entry of ribosomes into an internal ribosomal entry site (IRES) at the 5 nontranslated region. A region encoding the N-terminal part of the HCV polyprotein has been shown to augment the translation of HCV mRNA. Here we show that a cellular protein, NS1-associated protein 1 (NSAP1), augments HCV mRNA translation through a specific interaction with an adenosine-rich protein-coding region within the HCV mRNA. The overexpression of NSAP1 specifically enhanced HCV IRES-dependent translation, and knockdown of NSAP1 by use of a small interfering RNA specifically inhibited the translation of HCV mRNA. An HCV replicon RNA capable of mimicking the HCV proliferation process in host cells was further used to confirm that NSAP1 enhances the translation of HCV mRNA. These results suggest the existence of a novel mechanism of translational enhancement that acts through the interaction of an RNA-binding protein with a protein coding sequence.The translation of eukaryotic mRNAs occurs either by capdependent scanning or by direct binding of a ribosome to a specialized RNA element called an internal ribosomal entry site (IRES) (12). Although the canonical translation initiation factors function in translation through IRES elements, many RNA-binding proteins have also been shown to play important roles (12,14). For instance, host cellular proteins such as polypyrimidine tract-binding protein (PTB), the La autoantigen, poly(rC)-binding proteins, and upstream of N-ras (Unr) bind directly to IRESs and enhance the translation of picornaviral mRNAs (12,14).Hepatitis C virus (HCV), the major causative agent of virusrelated liver cirrhosis and hepatocellular carcinoma in humans, is a positive-sense RNA virus. The HCV RNA contains a 341-nucleotide (nt) 5Ј nontranslated region (5ЈNTR) and a very long open reading frame encoding a polyprotein that is proteolytically processed into 10 or more viral proteins (9, 15). Translation of the HCV polyprotein is directed by an IRES element spanning the 5ЈNTR (29).Curiously, while most IRESs require only the 5ЈNTR for full activity, the HCV IRES depends on the presence of a proteincoding sequence downstream of the initiating AUG (19,26). An HCV RNA encoding the N-terminal part of the HCV polyprotein was shown to be required for full HCV IRES activity when heterologous reporter genes (influenza NSЈ and secreted alkaline phosphatase) were connected to the HCV 5ЈNTR (26). Moreover, the same region was absolutely required for the generation of a viable chimeric poliovirus that used the HCV IRES element for translation (19). However, several other reports have suggested that the core-coding sequence is not strictly essential for HCV IRES activity (27,29,30). Thus, the true molecular basis of translational activation by the HCV core-coding sequence is still a subject of some debate.Here we investigated the HCV IRES translational initiation mechanism by analyzing proteins that were found to specifically interact with the IRES element....

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

“…By using an mRNA that bears both the HCV 59UTR and HCV 39UTR as an affinity matrix, we specifically purified proteins that have been shown to interact with either the HCV 59UTR or HCV 39UTR, or both. The proteins IGF2BP1, hnRNP C, hnRNP D, hnRNP Q, polypyrimidine tract binding protein 1, PAI-1 mRNA binding protein 1, nucleolin, NF90/NFAR, RNA helicase A, and five subunits of eIF 3 (out of nine identified) match with proteins isolated from cytoplasmic Huh7 cell extract employing an HCV 59UTR that lacks domain I and represents the HCV IRES consisting of domains II to IV (Kim et al 2004;Lu et al 2004). HnRNP C, YB-1, polypyrimidine tract binding protein 1, DDX3, NF90/NFAR, and RNA helicase A have been identified as HCV 39UTR binding proteins in Huh7 extract (Gontarek et al 1999;Harris et al 2006;Isken et al 2007).…”

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

“…In the case of HCV, several RNA-binding proteins, including La, NS1-associated protein 1 (NSAP1), and polypyrimidine tractbinding protein (PTB), have been shown to bind to the 5¢-end of HCV RNA and regulate HCV internal ribosome entry site (IRES)-mediated translation [5][6][7]. In addition, several cellular proteins have been shown to bind HCV nonstructural (NS) proteins; for example, human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33) was reported to bind to NS5A and NS5B proteins, and recruited them to the membrane to form the HCV replication complex (RC) [8,9].…”

New insights into internal ribosome entry site elements relevant for viral gene expression

“…Novel picornavirus IRES-interacting factors have been recently identified by RNA chromatography methods and mass spectrometry analysis (Bedard et al, 2007;Kim et al, 2004;Choi et al, 2004). Some of these proteins have been shown to act as activators or repressors of IRES activity.…”