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

Abstract

Translation of hepatitis C virus (HCV) 1 is mediated by an internal ribosome entry site (IRES) located mostly within the 5Ј-untranslated region (UTR) and extending a few nucleotides into the open reading frame (1-5). HCV 5Ј-UTR is highly conserved and folds into a complex secondary structure comprising four major structural domains (I-IV) and a pseudoknot in the vicinity of the initiator AUG codon (6, 7). The cis-elements promote assembly of initiation complex independent of the 5Ј-end and thus mediate internal initiation of translation in a cap-independent manner (7). Domain I is not a part of IRES and most likely is involved in RNA replication, whereas domains II and III are complex and consist of multiple stem-loops and bulge-loops. Even minor mutations in domains II and III substantially reduce IRES activity, but this could in most cases be regained by compensatory second site mutations that restored secondary structure. Highly conserved residues are often unpaired and may thus be able to interact with the components of the translation apparatus. Domain IV consists of a stem-loop that contains initiator AUG codon and has been shown to play a key role in regulating the initiation of translation of the HCV RNA (5, 8 -10). It appears from earlier reports that both the sequence and stability of the domain IV stem might control efficiency of HCV IRES translation (11, 12). These observations have led to a model for IRES function in which the structural elements in the IRES act as a scaffold that orients the potential binding sites in such a way that their interactions with initiation factors and ribosomes lead to assembly of functional ribosomal initiation complexes (13).HCV IRES binds to the 40 S ribosomal subunit specifically and stably even in the absence of any initiation factors. Addition of eIF2/GTP/Met-tRNAi is sufficient for 40 S subunit to lock onto initiator AUG (13). eIF3, though not essential for the formation of 48 S complex formation, it has been shown to bind to the apical half of domain III and is likely to be a constituent of the 48 S-IRES complex in vivo (14,15). 48 S complex formation on HCV IRES has no requirement for eIF4A, 4B, 4E, 4G, or for ATP hydrolysis (14 -16). Because the viral 5Ј-UTR forms a binary complex with the 40 S ribosomal subunit in the absence of any canonical or non-canonical initiation factors, it is likely that the additional factors may stimulate internal initiation of translation following the assembly of RNA-40 S complex. 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). However, eukaryotic mRNAs and picornaviral IRESs have not been reported to require S5 protein for the ribosome asse...

The RNA-binding protein La contributes to cell proliferation and CCND1 expression

“…For instance, human La supports internal ribosome entry site (IRES)-and cap-dependent translation of viral mRNAs, such as hepatitis C virus, polio virus (Ali et al, 2000;Costa-Mattioli et al, 2004;Pudi et al, 2004), HIV TAR mRNA (Svitkin et al, 1994;Chang et al, 1995), suppresses translation of the hepatitis A virus (Cordes et al, 2008), and regulates IRES-and cap-dependent translation of cellular mRNAs (for example, BiP mRNA , X-linked inhibitor of apoptosis protein (Holcik and Korneluk, 2000), 5 0 TOP-mRNAs (Crosio et al, 2000) and Mdm2 mRNA (Trotta et al, 2003)). …”

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

“…However, the scope of an siRNA-/shRNA-based antiviral strategy in general is limited because of the evolution of escape mutants. In this study, we purposely targeted the site (nt 331-350) within the SLIV region which encompasses cis-acting elements critical for ribosome assembly and which is likely to be more conserved in the HCV RNA and is protected from the generation of escape variants (Pudi et al, 2004). Thus, the sh-SLIV could be used to achieve broad spectrum inhibition of HCV translation and replication.…”

“…Previously, we have demonstrated the importance of the GCAC sequence near the iAUG for ribosome assembly onto HCV IRES RNA (Pudi et al, 2003(Pudi et al, , 2005. In fact, human La protein (an important host factor) has been shown to bind to the above sites and a peptide derived from the RNA-binding region of La protein has been shown to interfere with the ribosome assembly and inhibit viral RNA translation (Ali et al, 2000;Mondal et al, 2008;Pudi et al, 2004Pudi et al, , 2005. Here, we have investigated the ability of an shRNA targeting this region of HCV IRES to inhibit translation and replication of viral RNA.…”

The RNA chaperon activity of the human La protein (LARP3)

“…However, not much is known about the molecular mechanism by which La facilitates or impairs protein synthesis. Previous work suggested that the La protein might facilitate 48S complex formation during translation initiation of polio virus and hepatitis C virus translation and that La might bind in close proximity to translation start sites [31][32][33][34] . Furthermore, earlier work proposed that the C-terminal domain of La is required to promote polio virus IRES-mediated translation [35] .…”