The human La autoantigen has been shown to interact with the internal ribosome entry site (IRES) of hepatitis C virus (HCV) in vitro. Using a yeast three-hybrid system, we demonstrated that, in addition to full-length La protein, both N-and C-terminal halves were able to interact with HCV IRES in vivo. The exogenous addition of purified full-length and truncated La proteins in rabbit reticulocyte lysate showed dose-dependent stimulation of HCV IRES-mediated translation. However, an additive effect was achieved adding the terminal halves together in the reaction, suggesting that both might play critical roles in achieving full stimulatory activity of the full-length La protein. Using computational analysis, three-dimensional structures of the RNA recognition motifs (RRM) of the La protein were independently modeled. Of the three putative RRMs, RRM2 was predicted to have a good binding pocket for the interaction with the HCV IRES around the GCAC motif near the initiator AUG and RRM3 binds perhaps in a different location. This observation was further investigated by the filterbinding and toe-printing assays. The results presented here strongly suggest that both the N-and C-terminal halves can interact independently with the HCV IRES and are involved in stimulating internal initiation of translation. Hepatitis C virus (HCV)1 is a single-stranded, positive-sense enveloped RNA virus classified in a separate genus of the family Flaviviridae (1). HCV has been shown to be the primary causative agent of non-A, non-B viral hepatitis, which often leads to development of chronic hepatitis, cirrhosis, or hepatocellular carcinoma (2, 3). The HCV genome RNA is ϳ9.5 kb in length and consists of a 5Ј-untranslated region (UTR), a long open reading frame encoding the viral polyprotein, and a 3Ј-UTR (4). The translation initiation of HCV occurs by capindependent mechanism that is directed by the highly structured 5Ј-UTR (341 nt long), consisting of four stem-loops and a pseudoknot structure. The 5Ј-UTR contains unique cis-acting element called "internal ribosome entry site" (IRES) that mediates 5Ј-cap-independent internal initiation of translation (5-9). The HCV IRES element requires most of the 5Ј-UTR, except the first 40 nt, and extends to a short stretch (30 -40 nt) of sequence downstream of the initiator AUG codon (6, 10). Hepatitis C virus IRES is significantly different from that of picornaviruses (e.g. poliovirus) with respect to length, secondary and tertiary structure of the 5Ј-UTR RNA (11). Also in contrast to picornavirus IRES, HCV IRES does not have a strict requirement for the canonical initiation factors (eIFs) other than eIF2 and eIF3 (12). A number of cellular polypeptides have been shown to specifically interact with the HCV 5Ј-UTR RNA, which include polypyrimidine-tract binding protein (p57/60) (13), human La antigen (La, p50/52) (14), poly(rC)-binding protein 2 (15), heterogeneous nuclear ribonucleoprotein L (p68) (16), and ribosomal protein factors S9 and S5 (12). Interaction of these proteins with the 5Ј-UTR may...

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...

Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.Hepatitis C virus (HCV), a member of the Flaviviridae family, is an enveloped positive-sense, single-stranded RNA virus (10). The 9.6-kb-long genome encodes a single polyprotein of about 3,000 amino acids. The polyprotein is processed by host cell and viral proteases into three major structural proteins and several nonstructural proteins necessary for viral replication (4, 18). HCV causes a variety of liver diseases in humans, including liver cirrhosis and hepatocellular carcinoma (20,32). It is estimated that about 3% of the world population is infected with HCV, and about 85% of infected individuals develop chronic infection. Current options for treating HCV involve alpha interferon alone or in combination with ribavirin (6, 13). However, these treatments fail to achieve sustained virological response in the majority of patients, thus emphasizing the need for novel therapeutic approaches to combat HCV infection.Translation initiation of HCV occurs in a cap-independent manner wherein the ribosomes are recruited onto an internal ribosome entry site (IRES) located mostly within the 5Ј untranslated region (UTR) and extending a few nucleotides into the coding region (31, 37, 39). HCV IRES has been shown to form three complex stem-loops and a pseudoknot, which encompasses the initiator AUG codon (7). Although the HCV IRES binds to the 40S ribosomal subunit specifically and stably even in absence of any initiation factors, efficient translation requires canonical initiation factors like eIF2 and eIF3 (21,27,33) and other noncanonical trans-acting cellular proteins including polypyrimidine tract-binding protein (1), La autoantigen (2), poly(rC) binding protein (1...

Translation initiation of hepatitis C virus RNA occurs via ribosome binding to an 'internal ribosome entry site (IRES)' located in the 5'untranslated region of the viral RNA. The principle interaction between the 40S ribosomal subunit and the HCV IRES has been shown to be largely factor independent, which is followed by the joining of the 60S ribosomal subunit to form functional 80S complex. However several additional cellular proteins have been reported to bind to HCV IRES and enhance the initiation of translation. In order to identify novel factors involved in the ribosome assembly during internal initiation of HCV RNA, northwestern screening of a HeLa cDNA expression library was performed, using HCV IRES RNA as probe. We demonstrate here, that human ribosomal protein L18a, a constituent of 60S subunit, interacts with HCV IRES RNA. This interaction was further confirmed by using a recombinant protein similar to L18a (sL18a), cloned from human blood. Interestingly, addition of increasing concentration of the purified recombinant sL18a protein, showed moderate stimulation of HCV IRES activity in the in vitro translation assay. These observations suggest that the human L18a might influence the HCV IRES mediated translation.

Rinderpest virus (RPV) is an important member of the Morbillivirus genus in the family Paramyxoviridae and employs a similar strategy for transcription and replication of its genome as that of other negative sense RNA viruses. Cellular proteins have earlier been shown to stimulate viral RNA synthesis by isolated nucleocapsids from purified virus or from virus-infected cells. In the present work, we show that plus sense leader RNA of RPV, transcribed from 3' end of genomic RNA, specifically interacts with cellular La protein employing gel mobility shift assay as well as UV cross-linking of leader RNA with La protein. The leader RNA synthesized in virus-infected cells was shown to interact with La protein by immunoprecipitation of leader RNA bound to La protein and detecting the leader RNA in the immunoprecipitate by Northern hybridization with labeled antisense leader RNA. Employing a minireplicon system, we demonstrate that transiently expressed La protein enhances the replication/transcription of the RPV minigenome in cells. Sub-cellular immunolocalization shows that La protein is redistributed from nucleus to the cytoplasm upon infection. Our results strongly suggest that La protein may be involved in regulation of Rinderpest virus replication.