Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation.

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

4 antigen contains three putative RNA recognition motifs (RRM) and a basic region followed by a stretch of acidic region at the C-terminus (26). The C-terminus also contains a homodimerization domain that is required for the function of La in enhancing translation of poliovirus RNA (27). While a fraction of La antigen is found in the cytoplasm, majority of the protein is localized in the nucleus. However, cellular stress such as that resulting from a viral infection causes redistribution of the nuclear La to the cytoplasm (28). In the nucleus, La antigen transiently associates with the 3' oligo (U) terminus of the RNA polymerase III transcripts and facilitates transcription termination and recycling of transcription complexes for reinitiation process (29). Phosphorylation of Ser 366 residue of La antigen by casein kinase II modulates its role in transcription but does not affect its RNA binding ability (30). In the cytoplasm, La antigen is associated with a subset of small ribosomal subunit possibly by direct association with 18S rRNA (31). La protein has been shown to interact with the 5'NCR or 5' stem-loop structures of poliovirus (28), rubella virus (32), influenza virus (33), sindbis virus (34), the human immunodeficiency virus (HIV) TAR element (35), rhabdoviruses VSV (36) and rabies virus (37). For the most part, these La interactions with viral RNA elements do not appear to be mediated by the 3' terminal oligouridylate sequences that are characteristics of La protein's prototypical interactions with pre-processed polIII transcripts. RNA selection experiments with La protein have revealed a novel CACAA motif that may account for some non-protypical RNA interactions (38, DJK and JDK, manuscript in preparation). One RNA species, pre-tRNA met -elongator (termed C5 RNA by Harada et al., [39]) was found to have a unique relationship to La protein, as determined by its ability to bind deletion mutant La 22-408 that was unable to bind any polIII transcripts in HeLa cells extracts (38). The C5 RNA contains both 3' oligouridylates as well as the CACAA motif. The presence of a double binding motif likely accounts for the increased affinity of C5 RNA relative to other polIII transcripts.
Many of the viral RNAs that interact with La protein also contain close or exact matches by guest on April 26, 2019 http://www.jbc.org/ Downloaded from to the CACAA motif within their 5'NCR sequences, suggesting that this element may play a role in translational regulation by La protein.
The aberrant translation initiation of poliovirus IRES-mediated RNA is corrected in the presence of La antigen that is also accompanied by a modest stimulation of translation (28,40). La binding to the HIV TAR structure alleviates the translational repression exerted by TAR on the downstream reporter gene (41). We have recently shown that La antigen interacts with the HCV IRES which leads to significant level of stimulation of the HCV IRES-dependent translation (42). One of the interesting and novel aspects of this interaction is that it occurred in the context of the initiator AUG.
Here, we have further characterized cis-and trans-elements involved in the La-5'NCR interactions. The functional importance of these interactions during transactivation of the HCV IRES is investigated using mutants derived from the HCV 5'NCR as well as those in the La protein. An RNA SELEX ligand that specifically interacts with La protein inhibited the HCV IRES-mediated translation in a liver derived cell line and HeLa lysates supporting the in vivo functional relevance of that interaction. The data presented here further reinforces our previous observations that initiator AUG codon of the HCV RNA is an essential recognition motif of La binding. This interaction appears to be responsible for the La-dependent translation initiation of the HCV RNA.
Both the plasmids represent HCV 5'NCR sequence of NCR-C(AUG) except that they lack 120 and 135 nt from the 5' end of the NCR respectively. The plasmid NCR-C(AUG) was mutated at nt positions 126-129 (CUCC to AGAA) and 327-329 (CGU to GCA) of the HCV 5'NCR to construct pNCR-C(SI Mut) and pNCR-C(SII Mut) respectively. The plasmid NCR-C(Py-III Mut) was constructed by substitution of UUUCU to AAAGA at nt 194-198 in the pNCR-C(AUG). The nt sequence in these plasmids were confirmed by dideoxyribonucleotide sequencing method. The construction of His-tagged La encoding cDNA is described in detail by Goodier et al. (26). Selection of high affinity C5 RNA ligand against La antigen by SELEX method and construction of plasmid pC5-SP64 that expresses C5 RNA is described previously (38).
In vitro transcription-RNA transcripts were synthesized in vitro from linearized plasmid DNA that was purified by elution of the desired fragments from the agarose gels after digestion with an appropriate restriction endonuclease. The pNCR-C (AUG)] and all its derivatives were linearized with EcoRI and transcribed with T7 RNA polymerase using standard protocols. The plasmids pT7C1-341, (16)  Leupeptin) and stored in aliquote at -80 C. The glutathione-S-transferase (GST) and GST-PTB fusion protein were expressed in bacteria using plasmids GEX-KT and GST-2TK/PTB respectively, and purified as described earlier (43).
8 or lysates at 30 C for 30 min in RNA binding buffer (buffer D plus 2 mM MgCl 2 ) as described previously (42). For all the competition assays, competitor RNAs were added along with the components of the reaction mixture prior to UV cross-linking. The UV cross-linking was carried out in Stratalinker (Stratagen) for 30 min at 4 C. The ribonucleoprotein complexes were treated with RNase A (10-20 U) (USB) and analyzed by sodium dodecyl sulfate-polyacrylamide (12%) gel electrophoresis (SDS-PAGE) followed by autoradiography. The intensity of the bands were quantitated by PhosphorImager.
Immunoprecipitation of cellular La antigen-HCV 5'NCR complexes-HeLa S10 cytoplasmic fractions from suspension cultures of HeLa S3 were prepared essentially as described by Barton et al. (45). These lysates were active for the HCV IRES-mediated translation of reporter RNAs. Huh7 cells were cultured on petri dishes and the S10 lysates were prepared using the same method. S10 fraction (50 ug  A liver-derived cell line (Huh7) was used for the co-transfection assay. The cells were transfected with in vitro transcribed uncapped T7C1-341 or capped dicistronic dual luciferase RNA using lipofectin transfection protocol (Gibco BRL). To demonstrate the inhibitory activity of C5 SELEX or its mutant on the HCV IRES-controlled translation, the cells were directly co-transfected with the reporter RNAs along with C5 SELEX. An RNA derived from pGEM-4 multiple cloning site served as a control during a similar cotransfection experiment. Each transfection was carried out in triplicate. The luciferase activity was assayed as described above.

RESULTS
The C-terminus of La protein modulates its interaction with the HCV IRES-La antigen contains three putative RNA recognition motifs (RRMs). RRM1 and RRM2 are located at the N-terminal half of La protein and RRM3 is located at the beginning of the C-terminal portion ( Fig. 2A). Each of these RRMs play important role during direct recognition of the RNA motifs and increase the affinity of La during RNA binding by the other RRM (26). The C-terminus contains an adjacent basic and acidic region, phosphorylation sites and a homodimerization domain that regulate its nuclear and cytoplasmic functions (26,27,30). Using various La antigen mutants shown in Fig Multiple forms of cellular La antigen bind the HCV 5'NCR-Since the functional significance of La antigen interaction to the 5'NCR was investigated in both the liver (Huh7) and non-liver (HeLa) derived translation systems (see below), we first determined the presence of La protein in the cytoplasmic fractions (S10) of these cell lines. The S10 lysates were UV cross-linked with thio-U containing 32 P-labeled wild type HCV 5'NCR [NCR-C(AUG)] and immunoprecipitated using affinity purified human La antibodies (V3) (Fig. 5). Two closely migrating UV cross-linked 5'NCR-La complexes of the HeLa S10 at 50/52 kDa were observed following immunoprecipitation with the human La antibodies (lane 4) but not with the normal human IgG (NHIgG) (lane 3).
When Huh7 S10 lysates were used during this assay, a 43 kDa and a 50 kDa UV cross- The HCV IRES activity was specifically inhibited by the C5 RNA but not by the control RNA (unrelated) (Fig. 8A). The TMEV IRES which served as a negative control in our in vitro assay (Fig. 7D), was found to be inactive in Huh7 cells after repeated transfection studies. However, other controls such as poliovirus IRES was efficiently inhibited by the C5 SELEX (data not shown). A dual luciferase assay system was employed to further evaluate the specificity of C5 SELEX inhibitory activity. In vitro transcribed capped RL-1b contains upstream renilla luciferase (RL) that is translated by cap-dependent mechanism whereas the translation of downstream firefly luciferase (FL) is controlled by the HCV IRES (Fig 8B, upper panel). During transfection studies as described above, a dramatic inhibition of the HCV IRES in the context of the dual reporter RNA was observed by wild type (WT) C5 RNA (Fig. 8B) whereas the mutant (Mut) C5 exhibited moderate inhibitory activity similar to that seen during our in vitro analysis (Fig. 7E). Because of the fact that RRL contain comparatively little La protein than HeLa lysates, the effect of recombinant La was more pronounced in this system. Belsham et al. (49) attempted La-depletion strategy to demonstrate the functional importance of La protein during poliovirus IRES-mediated translation.
In that assay, La antigen was immunodepleted from HeLa lysate. Interestingly, the La-depletion dramatically abolished the translation ability of the lysate which was not rescued by the addition of purified La protein. The explanation was that such an approach suffers from the removal of La-associated factors that may be essential element(s) of translation initiation. A La-depletion/add-back approach was also employed by Das et al., (50) during their studies on the inhibition of the HCV IRES-mediated translation by yeast I-RNA that strongly binds human La antigen. In this experiment the translation lysates were passed through an I-RNA affinity column, and the flow through that was assayed for HCV IRES function failed to support translation. During an add-back experiment described here (Fig. 6), again the HCV IRES activity was rescued to the basal level that suggested the requirement of additional factor(s) for the stimulated translation. In light of the failure of some of these approaches, we sought an alternative strategy of employing SELEX RNA molecules with high affinity to La protein to establish the requirement of La antigen for the HCV IRES activity in vivo. We have utilized a high affinity RNA ligand , termed C5 SELEX RNA, that binds to the La antigen (Fig. 7A). This In a similar study, 4 subunits of the eIF-3 (p170, p116, p66 and p47) were also shown to make direct contact with the apical half of the domain III of the 5'NCR (52). In our studies, we observed that La antigen binding to the 5'NCR was unperturbed when increasing concentrations of 40S ribosomal subunit were added to the assay mixture (unpublished data). These and other studies suggest that the interactions of cellular proteins may be directed toward specific sites in the 5'NCR. Furthermore, eIF-3 and La antigen are known to associate with native 40S ribosomal subunit in the cytoplasm (1,31). Since structural integrity of the 5'NCR is important for its IRES function, it is highly likely that the structural elements of the HCV IRES provide a "docking facility" for the ribosomes. The ribosomal docking must be mediated by these cellular factors to direct the complex to the designated initiation site. In this scenario, La antigen, may be functioning as a molecular chaperon during assembly of the ribosomal complex directly at the initiation site.
Based on the inherent feature of La antigen for its ability to unwind the double stranded RNA in a ATP-dependent manner (24), it is conceivable that the La binding to the translation initiation site provides additional advantage for the initiation complex in acquiring RNA helicase activity. Interestingly, the flanking sequences of the initiator AUG form a stable stem-loop structure (53, Fig. 1