Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation Is Stimulated by Specific Interaction of Independent Regions of Human La Autoantigen

Abstract

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...

La protein binds the predicted loop structures in the 3′ non-coding region of Japanese encephalitis virus genome: role in virus replication

“…Many of the viral RNAs interacting with La protein also contain a close match or exact CACAA sequence (Ali et al, 2000). For example, La protein interacts with the HCV 59 NCR with a GCAC sequence (Pudi et al, 2003). The toe-printing experiments described here indicated that La protein interacted with sequence ACA (nt 10929-10931) in loop I of the JEV 39 SL RNA, whilst it bound to sequence AAG (nt 10940-10942) in loop II.…”

“…La protein contains three RRMs: RRM1 and RRM2 are located in the N-terminal half of the protein, whilst RRM3 is located in the C-terminal half. The N-terminal domain of La protein has been shown to interact with the poliovirus 59 NCR (Izumi et al, 2004;Svitkin et al, 1994), whilst the C-terminal domain interacts with the HCV 59 NCR in vitro (Ali et al, 2000); however, both Nand C-terminal halves are able to interact with the viral RNA independently in vivo (Pudi et al, 2003). We have shown that La protein interaction with the JEV 39 NCR involves its N-terminal half only.…”

“…Escherichia coli plasmids expressing human La protein or its truncated forms under the control of the bacteriophage T7 promoter were kindly provided by Dr S. Das, The Indian Institute of Science, Bangalore, India (Pudi et al, 2003). The recombinant protein was expressed in E. coli BL21(DE3) cells and purified using Ni-NTA agarose (Qiagen).…”

“…Construction of pJE3SL, containing cDNA for JEV 39 SL RNA, has been described previously (Ta & Vrati, 2000). The plasmid was linearized with XbaI and transcribed in vitro using T7 DNA polymerase by using the Riboprobe system (Promega) to produce 32 P-labelled JEV 39 SL RNA (Ta & Vrati, 2000).Synthesis of recombinant La protein and its truncated forms.Escherichia coli plasmids expressing human La protein or its truncated forms under the control of the bacteriophage T7 promoter were kindly provided by Dr S. Das, The Indian Institute of Science, Bangalore, India (Pudi et al, 2003). The recombinant protein was expressed in E. coli BL21(DE3) cells and purified using Ni-NTA agarose (Qiagen).…”

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

“…Since the mechanism of ribosome assembly is unique and fundamentally different from the cap-dependent translation of the host cell mRNA, it can be targeted to selectively inhibit viral protein synthesis and consequently its replication (Dasgupta et al, 2004;Hellen & Sarnow, 2001;Trowbridge & Gowans, 1998). 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.…”