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• W2144195875 abstract "Human La autoantigen has been shown to influence internal initiation of translation of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that, among the three RRMs of La protein, the RRM2 interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV (SL IV) (Pudi, R., Abhiman, S., Srinivasan, N., and Das S. (2003) J. Biol. Chem. 278, 12231–12240). Here, we have demonstrated that the mutations in the GCAC motif, which altered the binding to RRM2, had drastic effect on HCV IRES-mediated translation, both in vitro and in vivo. The results indicated that the primary sequence of the stem region of SL IV plays an important role in mediating internal initiation. Furthermore, we have shown that the mutations also altered the ability to bind to ribosomal protein S5 (p25), through which 40 S ribosomal subunit is known to contact the HCV IRES RNA. Interestingly, binding of La protein to SL IV region induced significant changes in the circular dichroism spectra of the HCV RNA indicating conformational alterations that might assist correct positioning of the initiation complex. Finally, the ribosome assembly analysis using sucrose gradient centrifugation implied that the mutations within SL IV of HCV IRES impair the formation of functional ribosomal complexes. These observations strongly support the hypothesis that La protein binding near the initiator AUG facilitates the interactions with ribosomal protein S5 and 48 S ribosomal assembly and influences the formation of functional initiation complex on the HCV IRES RNA to mediate efficient internal initiation of translation. Human La autoantigen has been shown to influence internal initiation of translation of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that, among the three RRMs of La protein, the RRM2 interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV (SL IV) (Pudi, R., Abhiman, S., Srinivasan, N., and Das S. (2003) J. Biol. Chem. 278, 12231–12240). Here, we have demonstrated that the mutations in the GCAC motif, which altered the binding to RRM2, had drastic effect on HCV IRES-mediated translation, both in vitro and in vivo. The results indicated that the primary sequence of the stem region of SL IV plays an important role in mediating internal initiation. Furthermore, we have shown that the mutations also altered the ability to bind to ribosomal protein S5 (p25), through which 40 S ribosomal subunit is known to contact the HCV IRES RNA. Interestingly, binding of La protein to SL IV region induced significant changes in the circular dichroism spectra of the HCV RNA indicating conformational alterations that might assist correct positioning of the initiation complex. Finally, the ribosome assembly analysis using sucrose gradient centrifugation implied that the mutations within SL IV of HCV IRES impair the formation of functional ribosomal complexes. These observations strongly support the hypothesis that La protein binding near the initiator AUG facilitates the interactions with ribosomal protein S5 and 48 S ribosomal assembly and influences the formation of functional initiation complex on the HCV IRES RNA to mediate efficient internal initiation of translation. Translation of hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; UTR, untranslated region; IRES, internal ribosome entry site; La, human La antigen; RRM, RNA recognition motif; nt, nucleotide(s); eIF, eukaryotic initiation factor; PTB, polypyrimidine tract-binding protein; PCBP, poly-r(C)-binding protein; SL, stem-loop; RRL, rabbit reticulocyte lysate; RLuc, Renilla luciferase; FLuc, firefly luciferase; CD, circular dichroism; rpS5, ribosomal protein S5; DTT, dithiothreitol; MEM, minimal essential medium; I-RNA, inhibitor RNA.1The abbreviations used are: HCV, hepatitis C virus; UTR, untranslated region; IRES, internal ribosome entry site; La, human La antigen; RRM, RNA recognition motif; nt, nucleotide(s); eIF, eukaryotic initiation factor; PTB, polypyrimidine tract-binding protein; PCBP, poly-r(C)-binding protein; SL, stem-loop; RRL, rabbit reticulocyte lysate; RLuc, Renilla luciferase; FLuc, firefly luciferase; CD, circular dichroism; rpS5, ribosomal protein S5; DTT, dithiothreitol; MEM, minimal essential medium; I-RNA, inhibitor RNA. is mediated by an internal ribosome entry site (IRES) located mostly within the 5′-untranslated region (UTR) and extending a few nucleotides into the open reading frame (1Tsukiyama-Kohara Z. Iizuka N. Kohara M. Nomoto A. J. Virol. 1992; 66: 1476-1483Crossref PubMed Google Scholar, 2Wang C. Sarnow P. Siddiqui A. J. Virol. 1993; 67: 3338-3344Crossref PubMed Google Scholar, 3Reynolds J.E. Kaminski A. Carroll A.R. Clarke B.E. Rowlands D.J. Jackson R.J. RNA (N. Y.). 1996; 2: 867-878PubMed Google Scholar, 4Lu H.-H. Wimmer E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1412-1417Crossref PubMed Scopus (152) Google Scholar, 5Kamoshita N. Tsukiyama-Kohara K. Kohara M. Nomoto A. Virology. 1997; 233: 9-18Crossref PubMed Scopus (77) Google Scholar). HCV 5′-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 (6Brown E.A. Zhang H. Ping L. Lemon S.M. Nucleic Acids Res. 1992; 20: 5041-5045Crossref PubMed Scopus (352) Google Scholar, 7Rijnbrand R.C.A. Lemon S.M. Current Topics in Microbiology and Immunology. Springer-Verlag, Berlin, Germany2000: 85-111Google Scholar). The cis-elements promote assembly of initiation complex independent of the 5′-end and thus mediate internal initiation of translation in a cap-independent manner (7Rijnbrand R.C.A. Lemon S.M. Current Topics in Microbiology and Immunology. Springer-Verlag, Berlin, Germany2000: 85-111Google Scholar). 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 (5Kamoshita N. Tsukiyama-Kohara K. Kohara M. Nomoto A. Virology. 1997; 233: 9-18Crossref PubMed Scopus (77) Google Scholar, 8Honda M. Ping L.H. Rijnbrand R.C.A. Amphlett E. Clarke B. Rowlands D. Lemon S.M. Virology. 1996; 222: 31-42Crossref PubMed Scopus (216) Google Scholar, 9Rijnbrand R. Bredenbeek P. van der Straaten T. Whetter L. Inchauspe G. Lemon S. Spaan W. FEBS Lett. 1995; 365: 115-119Crossref PubMed Scopus (156) Google Scholar, 10Lemon S.M. Honda M Semin. Virol. 1997; 8: 274-288Crossref Scopus (70) Google Scholar). It appears from earlier reports that both the sequence and stability of the domain IV stem might control efficiency of HCV IRES translation (11Honda M. Brown E.A. Lemon S.M. RNA (N. Y.). 1996; 2: 955-968PubMed Google Scholar, 12McKnight K.L. Sandefur S. Phipps K.M. Heinz B. Virology. 2003; 317: 345-358Crossref PubMed Scopus (1) Google Scholar). 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 (13Hellen C.U.T. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (802) Google Scholar). 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 (13Hellen C.U.T. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (802) Google Scholar). 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 (14Pestova T.V. Shatsky I.N. Fletcher S.P. Jackson R.J. Hellen C.U. Genes Dev. 1998; 12: 67-83Crossref PubMed Scopus (626) Google Scholar, 15Sizova D.V. Kolupaeva V.G. Pestova T.V. Shatsky I.N. Hellen C.U.T. J. Virol. 1998; 72: 4775-4782Crossref PubMed Google Scholar). 48 S complex formation on HCV IRES has no requirement for eIF4A, 4B, 4E, 4G, or for ATP hydrolysis (14Pestova T.V. Shatsky I.N. Fletcher S.P. Jackson R.J. Hellen C.U. Genes Dev. 1998; 12: 67-83Crossref PubMed Scopus (626) Google Scholar, 15Sizova D.V. Kolupaeva V.G. Pestova T.V. Shatsky I.N. Hellen C.U.T. J. Virol. 1998; 72: 4775-4782Crossref PubMed Google Scholar, 16Pestova T.V. Hellen C.U.T. Virology. 1999; 258: 249-256Crossref PubMed Scopus (59) Google Scholar). Because the viral 5′-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 (14Pestova T.V. Shatsky I.N. Fletcher S.P. Jackson R.J. Hellen C.U. Genes Dev. 1998; 12: 67-83Crossref PubMed Scopus (626) Google Scholar, 17Fukushi S. Kurihara C. Ishiyama N. Hoshino F.B. Oya A. Katayama K. J. Virol. 1997; 71: 1662-1666Crossref PubMed Google Scholar, 18Fukushi S. Okada M. Stahl J. Kageyama T. Hoshino F.B. Katayama K. J. Biol. Chem. 2001; 276: 20824-20826Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). 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 (14Pestova T.V. Shatsky I.N. Fletcher S.P. Jackson R.J. Hellen C.U. Genes Dev. 1998; 12: 67-83Crossref PubMed Scopus (626) Google Scholar). However, eukaryotic mRNAs and picornaviral IRESs have not been reported to require S5 protein for the ribosome assembly. Recently we have shown that a small RNA corresponding to the stem-loop III e+f domain of the HCV IRES, when introduced in trans, can antagonize cellular protein binding to the viral IRES and inhibits HCV IRES-mediated translation. The RNA molecule showed strong interaction with the ribosomal S5 protein and prevented the recruitment of the 40 S ribosomal subunit by the HCV IRES (19Ray P.S. Das S. Nucleic Acids Res. 2004; 32: 1678-1687Crossref PubMed Scopus (31) Google Scholar). Therefore, it appears that any event that might facilitate S5 interaction with HCV IRES could be crucial for efficient ribosome assembly at the initiation site. It is believed that HCV IRES contains one set of determinants that is required for initial ribosome recruitment and a second set that promotes accurate placement of the initiation codon in the ribosomal P site (13Hellen C.U.T. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (802) Google Scholar). Several cellular trans-acting factors that are known to bind to HCV IRES and influence the internal initiation might play a role in the formation of functional initiation complex after the initial binding of 40 S ribosomes to the IRES. Most notable among these are the polypyrimidine tract-binding protein (PTB), (20Ali N. Siddiqui A. J. Virol. 1995; 69: 6367-6375Crossref PubMed Google Scholar) the La autoantigen (21Ali N. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2249-2254Crossref PubMed Scopus (246) Google Scholar), and poly(rC) binding protein 2 (PCBP 2) (22Fukushi S. Okada M. Kageyama T. Hoshino F.B. Nagai K. Katayama K. Virus Res. 2001; 73: 67-79Crossref PubMed Scopus (52) Google Scholar) etc. In case of picornaviruses, a model has been proposed in which the IRES trans-acting factors such as PTB help the RNA to attain or maintain an active conformation in which it is able to bind essential factors and the 43 S ribosome complex in a productive manner (23Kaminski A. Jackson R.J. RNA (N. Y.). 1998; 4: 626-638Crossref PubMed Scopus (114) Google Scholar). In hepatitis A virus, glyceraldehyde-3-phosphate dehydrogenase was found to destabilize the folded structures of the RNA stem-loops and influence the IRES activity (24Schultz D.E. Hardin C.C. Lemon S.M. J. Biol. Chem. 1996; 271: 14134-14142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Human La autoantigen was found to enhance the accuracy of initiation codon selection in poliovirus IRES (25Meerovitch K. Svitkin Y.V. Lee H.S. Lejbkowicz F. Kenan D.J. Chan E.K. Agol V.I. Keene J.D. Sonenberg N. J. Virol. 1993; 67: 3798-3807Crossref PubMed Google Scholar). The human La protein is an RNA-binding protein, belonging to the RNA recognition motif (RRM) superfamily (26Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 841-850Crossref PubMed Scopus (174) Google Scholar, 27Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 851-861Crossref PubMed Scopus (301) Google Scholar). La protein is ubiquitously expressed in many eukaryotic organisms, including Saccharomyces cerevisiae, Xenopus laevis, and Drosophila melanogaster (28Yoo C.J. Wolin S.L. Mol. Cell. Biol. 1994; 14: 5412-5424Crossref PubMed Scopus (106) Google Scholar). La protein has been shown to interact with a wide variety of cellular and viral RNAs and has been implicated in various cellular processes, which include RNA polymerase III transcription termination (27Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 851-861Crossref PubMed Scopus (301) Google Scholar), telomere homeostasis (29Ford L.P. Sway J.W. Wright W.E. RNA (N. Y.). 2001; 7: 1068-1075Crossref PubMed Scopus (46) Google Scholar), internal initiation of translation of Bip mRNA, poliovirus, coxsackievirus B3, encephalomyocarditis virus, and hepatitis C virus (30Ray P.S. Das S. Nucleic Acids Res. 2002; 30: 4500-4508Crossref PubMed Google Scholar, 31Wolin S.L. Cedervall T. Annu. Rev. Biochem. 2002; 71: 375-403Crossref PubMed Scopus (333) Google Scholar). Also, La protein has been shown to be capable of unwinding DNA-RNA hybrids and double-stranded RNA in an ATP-dependent manner (32Huhn P. Pruijn G.J. van Venrooij W.J. Bachmann M. Nucleic Acids Res. 1997; 25: 410-416Crossref PubMed Scopus (58) Google Scholar). La protein specifically interacts with both the 5′- and 3′-UTR of HCV RNA (33Spangberg K. Wiklund L. Schwartz S. J. Gen. Virol. 2001; 82: 113-120Crossref PubMed Scopus (58) Google Scholar). La protein plays a functional role in internal initiation of translation where addition of purified La to RRL in the in vitro translation assays using HCV IRES resulted in stimulation of translation activity (21Ali N. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2249-2254Crossref PubMed Scopus (246) Google Scholar). Inhibition of HCV IRES activity caused by sequestration of La protein can be rescued by exogenous addition of purified La protein (34Das S. Ott M. Yamane A. Tsai W. Gromier M. Lahser F. Gupta S. Dasgupta A. J. Virol. 1998; 72: 5638-5647Crossref PubMed Google Scholar, 35Izumi R.E. Das S. Barat B. Raychaudhuri S. Dasgupta A. J. Virol. 2004; 78: 3763-3776Crossref PubMed Scopus (39) Google Scholar). Previously, La protein has been shown to bind to HCV IRES in the context of initiator AUG (21Ali N. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2249-2254Crossref PubMed Scopus (246) Google Scholar). Recently, we showed that, of the three RRMs present in La, RRM2 binds HCV RNA around the region encompassing the GCAC motif located in the stem region near the initiator AUG (36Pudi R. Abhiman S. Srinivasan N. Das S. J. Biol. Chem. 2003; 278: 12231-12240Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). However, the precise mechanism with which the La protein influences the stimulation of HCV IRES activity is yet to be elucidated. Here we report for the first time a possible functional role of La protein interaction near the initiator AUG of the HCV-IRES RNA to mediate efficient internal initiation of translation. We have demonstrated that mutations in the GCAC motif near initiator AUG within the SL IV, which alter the primary sequence while retaining the overall secondary structure affect the binding of La-RRM2 to HCV IRES. Also, these mutations have drastic effect on the HCV IRES-mediated translation both in vitro and in vivo, indicating that the sequence GCAC might play an important role in maintaining the IRES function. Further, we have observed that the mutations also alter the binding of certain cellular proteins to HCV IRES, especially p25 (S5), whose binding may have been influenced by the binding of La protein to HCV IRES in the SL IV region. Interestingly, addition of increasing concentration of La protein helped in the binding of S5 protein suggesting that La protein might play a role in recruitment of the 40 S ribosomal subunit to the HCV IRES RNA. Additionally, the circular dichroism spectra of HCV stem-loop IV RNA showed a dose-dependent increase in the presence of increasing concentrations of La protein indicating that HCV IRES may undergo some conformational alterations upon binding to La protein, which assist in the formation of functional initiation complex. Finally, sucrose gradient centrifugation analysis of ribosome assembly implied that the mutations within HCV IRES lead to reduced efficiency in the formation of functional ribosomal complex. Taken together, the results strongly suggest that La protein interaction near the initiator AUG might be involved in conformational alteration to facilitate better contact with the 40 S ribosomal subunit required for efficient internal initiation of translation. Plasmids—HCV 1b-encoding plasmid, pCV, was generously gifted by Dr. Akio Nomoto and Dr. Tsukiyama-Kohara, University of Tokyo, Japan. HCV 5′-UTR along with 42-nt coding sequence (18–383 nt) was cloned into the mammalian expression vector pcDNA3 (Invitrogen) to generate pcDHCV-383 as described earlier (36Pudi R. Abhiman S. Srinivasan N. Das S. J. Biol. Chem. 2003; 278: 12231-12240Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). pcD-SL IV containing nt 293–383 of HCV IRES was obtained by amplifying HCV SL IV using the primers, 5′-ATAGAAGCTTGCCTGATAGGTCTGCGA-3′ and 5′-CGCGAATTCGTTACGTTTGGTTTT-3′, from the template plasmid pc-DHCV-383 and cloned between HindIII and EcoRI sites of the pcDNA3. The PCRs were carried out with 30 cycles, each cycle consisting of denaturation (95 °C for 40 s), annealing (55 °C for 1 min), and extension (68 °C for 1 min/1 kb) using Pfx DNA polymerase (Invitrogen). The cDNA clone encoding human La autoantigen, pET-La was obtained from Dr. Jack Keene, Duke University. La coding sequence and La 101–208 (RRM2) were subcloned into pRSET-A vector (Invitrogen) (36Pudi R. Abhiman S. Srinivasan N. Das S. J. Biol. Chem. 2003; 278: 12231-12240Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Ribosomal protein S5 containing plasmid pQE-RS5 was obtained from Dr. Shuetsu Fukushi, Biomedical Laboratories, Japan. S5 coding sequence was obtained from the vector by digesting with HindIII and BamHI and subcloned into pRSET-A vector. All the mutants described in the study were generated by using a megapriming PCR method as described previously (37Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). The method utilizes three oligonucleotide primers to perform two rounds of PCR. The product of the first PCR is used as one of the primers (a “megaprimer”) for the second polymerase chain reaction. The first round of PCR was carried out using the 3′ primer corresponding to 383 nt of HCV RNA, 5′-CGCGAATTCGTTACGTTTGGTTTT-3′, and the following 5′ primers carrying the specific mutations, 5′-CCGTGCATCAAGAGCACAAAT-3′, 5′-TCGTAGACCACTAATCATGATAGTAAATCCTAA-3′, and 5′-TCGTAGACCCGGTATCATGAACCGAAATCCTAA-3′. The PCRs were carried out for 35 cycles, each cycle consisting of denaturation (95 °C for 40 s), annealing (55 °C for 1 min), and extension (72 °C for 10 s) using TaqDNA polymerase (Bangalore Genie) or Pfx polymerase. The amplification products were subjected to 9% native PAGE and visualized by staining with ethidium bromide. The bands were cut and incubated in 400 μl of elution buffer (0.5 m ammonium acetate, 10 mm magnesium acetate, 0.1% SDS) overnight at 37 °C. The supernatant was subjected to phenol-chloroform extraction, and the DNAs were alcohol-precipitated. The megaprimers thus obtained were then used as 3′ primers in the second round of PCR along with the HCV 5′ primer, 5′-TATAAGCTTGGATCCCCGGCGACA-3′. The PCRs were carried out for 35 cycles, each cycle consisting of denaturation (95 °C for 40 s), annealing (60 °C for 1 min), and extension (72 °C for 30 s) using TaqDNA polymerase or Pfx polymerase. The PCR products were digested with HindIII and EcoRI and cloned into pcDNA3 vector. pcD-SL IV M4mut was generated using the primers 5′-ATAGAAGCTTGCCTGATAGGGTGCTTGCGA-3′, 5′-CGCGAATTCGTTACGTTTGGTTTT-3′, using the plasmid M4 as template and cloned between HindIII and EcoRI sites of the pcDNA3. For the construction of bicistronic constructs, the two reporter genes, Renilla luciferase (RLuc) and Firefly luciferase (FLuc) coding sequences were PCR-amplified using Pfx polymerase using the following primers, 5′-GCATGCTAGCACCATGACTTCGAAA-3′, 5′-GGCCAAGCTTACCATGATTCGAAA-3′, 5-GCATCTCGAGGAAGACGCCAAAAAC-3′, and 5′-ATTAGGGCCCTTACACGGCGATCTT-3′. The 5′ primer of FLuc lacks the initiator AUG codon, because the HCV IRES has an initiator AUG located within. The amplified RLuc was cloned into NheI and XhoI sites of pcDNA3.1 (+) vector (Invitrogen), and FLuc was cloned into XhoI and ApaI sites. The wild-type and mutant HCV IRES were cloned into HindIII and EcoRI sites between the two reporter genes. Preparation of HeLa S10 Cell Extract—HeLa cells used for preparing cell extract were grown in T75 flasks in minimal essential medium (pH 7.0) supplemented with 10% fetal calf serum. Monolayer of HeLa cells were harvested, pelleted down, and washed thrice with cold isotonic buffer (35 mm HEPES, pH 7.4, 146 mm NaCl, 11 mm glucose), resuspended in 1.5× packed cell volume of hypotonic buffer (10 mm HEPES, pH 7.4, 15 mm KCl, 1.5 mm magnesium acetate, and 6 mm β-mercaptoethanol) and then incubated on ice for 10 min. Cells were then transferred to a Down's homogenizer and disrupted by 50 strokes on ice (lysis was checked under microscope). The lysate was incubated in 1× incubation buffer for 10 min. Cytoplasmic extract (S10 supernatant) was isolated by centrifuging the lysate at 10,000 × g for 30 min at 4 °C. The supernatant was dialyzed for 4 h against 100 volumes of dialysis buffer and aliquoted into pre-chilled tubes. The aliquots of S10 extract were stored at –70 °C. Purification of rpS5, La Full-length, and RRM2 Proteins Using a Nickel-Nitrilotriacetic Acid-Agarose Column—Escherichia coli BL21(DE3) cells were transformed with pRSET-A vectors containing either the full-length or the deletion mutant of La or the full-length rpS5. Transformed colonies were inoculated into 100 ml of LB broth containing 75 μg/ml ampicillin and grown at 37 °C in an incubator shaker at 200 rpm speed until the OD660 reached 0.6. The cultures were induced with 0.6 mm isopropyl-1-thio-β-d-galactopyranoside and grown for further 4 h. The cells were pelleted and resuspended in 5 ml of lysis buffer (50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole). The extract was made by sonication. The above crude extracts were mixed with 500 μl of nickelnitrilotriacetic acid-agarose slurry (Qiagen) and kept for rocking at 4 °C for 4 h. The lysate was loaded onto a column, and the flow-through was discarded. The column was washed with 50 ml of wash buffer (50 mm NaH2PO4, 300 mm NaCl, 40 mm imidazole). The bound protein was eluted with 500 μl of elution buffer containing 500 mm imidazole. The eluted proteins were dialyzed at 4 °C for 4–6 h in 500 ml of dialysis buffer (50 mm Tris (pH 7.4), 100 mm KCl, 7 mm β-mercaptoethanol, 20% glycerol), aliquoted, and stored in –70 °C freezer. In Vitro Transcription—mRNAs were transcribed in vitro from different linearized plasmid constructs under T7 promoters in run-off transcription reactions. The HCV bicistronic constructs were linearized with PmeI downstream of firefly luciferase and used as templates for RNA synthesis. The linear DNA were electrophoresed on agarose gels and extracted by using Qiagen gel elution kit and capped bicistronic RNA were synthesized using Ribomax Large scale RNA production system-T7 (Promega) under standard conditions with addition of 5′ cap analog (Promega). Radiolabeled mRNAs were transcribed in vitro using T7 RNA polymerase (Promega) and [32P]uridine triphosphate (PerkinElmer Life Sciences). The pcDNA3 vectors containing wild-type or mutant HCV IRES were linearized with EcoRI, gel-eluted, and transcribed in vitro to generate the 32P-labeled RNA. The transcription reaction was carried out under standard conditions (Promega protocol) using 2.5 μg of linear template DNA at 37 °C for 1.5 h. After alcohol precipitation, the RNA was resuspended in 25 μl of nuclease-free water. 1 μl of the radiolabeled RNA sample was spotted onto DE81 filter paper, washed with phosphate buffer, and dried, and the incorporated radioactivity was measured using liquid scintillation counter. Filter Binding Assay—The 32P-labeled wild-type or mutant HCV IRES RNAs were incubated with the La RRM2 or RRM3 proteins at 30 °C for 15 min in RNA binding buffer (containing 5 mm HEPES, pH 7.6, 25 mm KCl, 2 mm MgCl2, 3.8% glycerol, 2 mm DTT, and 0.1 mm EDTA) and loaded onto nitrocellulose filters equilibrated with the 2-ml RNA binding buffer. The filters were then washed four times with 1 ml of binding buffer and air-dried. The counts retained were measured in liquid scintillation counter. The graph was plotted with protein concentration (micromolar) on the x-axis, and the percentage of bound RNA was plotted as the percentage of counts retained, on the y-axis. UV Cross-linking Experiment—The 32P-labeled wild-type or mutant HCV IRES RNAs were incubated with the purified proteins or HeLa S10 extract at 30 °C for 15 min in RNA binding buffer (containing 5 mm HEPES, pH 7.6, 25 mm KCl, 2 mm MgCl2, 3.8% glycerol, 2 mm DTT, and 0.1 mm EDTA) and then irradiated with a hand-held UV lamp for 10 min. The mixture was treated with 30 μg of RNase A (Sigma) at 37 °C for 30 min. The protein-nucleotidyl complexes were electrophoresed on SDS-10% PAGE analyzed by phosphorimaging analysis. In the experiments performed in the presence of La protein, the RNA was incubated with the purified proteins at 30 °C for 10 min prior to binding with HeLa S10 extracts. In Vitro Translation—In vitro translation of the capped bicistronic mRNAs were carried out in micrococcal nuclease treated rabbit reticulocyte lysates (RRL, Promega Corp.). Briefly, 12.5-μl reaction mixtures contained 8.75 μl of RRL containing 0.25 μl each of minus methionine mixture, 5 μCi of [35S]methionine (PerkinElmer Life Sciences) and 10 units of RNasin (Promega) were incubated at 30 °C for 1.5 h. 1 μlofthe reaction mixtures was assayed for both the Renilla and firefly luciferase activity according to Promega protocol using Dual-Luciferase reporter assay system. 7 μl of the reaction mixtures was electrophoresed on SDS-10% polyacrylamide gel, dried, and analyzed by phosphorimaging. Transfection of HeLa/Huh7 Monolayer Cells—HeLa/Huh7 cells grown in 30-mm dishes at 60–70% confluence were transfected with 2 μg of the bicistronic constructs using Tfx 20 transfection reagent (Promega) according to manufacturer's protocol. Briefly, the DNA was mixed with 5 μl of Tfx 20 reagent and diluted to 1 ml using the MEM (Invitrogen) and incubated at room temperature for 15 min. The cells were washed with medium and overlaid with 1 ml of MEM containing the above DNAs. After 1 h of incubation at 37 °C, 0.8 ml of MEM, and 0.2 ml of fetal bovine serum (Invitrogen) was added to the dishes. After 36 h, the cells were washed with phosphate-buffered saline and lysed using 1× passive lysis buffer (Promega) and assayed for RLuc and FLuc according to Promega protocol using the Dual-Luciferase reporter assay system. CD Spectroscopy—Measurements of CD spectra were performed with a Jasco J-715 spectropolarimeter as described earlier (38Hardin C.C. Corregan M.J. Brown II, B.A. Frederick L. Biochemistry. 1993; 32: 5870-5880Crossref PubMed Scopus (65) Google Scholar). Spectra were obtained in 0.5 ml of RNA binding buffer (5 mm HEPES, pH 7.6, 25 mm KCl, 2 mm MgCl2, 3.8% glycerol, 2 mm DTT, 0.1 mm EDTA). CD spectra were obtained in the 240- to 320-nm range at 20 °C with HCV stem-loop IV WT or stem-loop IV M4 RNA (250 or 300 nm) and increasing concentration of purified recombinant La protein (0.5–2 μm). The molar ellipticity values were normalized for the contribution of the La protein at each concentration. Sucrose Gradient Centrifugation Analysis of Ribosomal Assembly on HCV IRES—The 32P-radiolabeled RNAs (∼2 × 105 cpm) were added to 50 μl of ribosome assembly reactions containing 35 μl of RRL, 0.5 μl each of minus methionine and minus leucine amino acid mixtures and 10 units of RNasin (Promega) and incubated at 30 °C for 15 min. The reactions were diluted to 150 μl with gradient buffer (20 mm Tris (pH 7.5), 100 mm KCl, 3 mm MgCl2, 1 mm DTT) and overlaid on a 5–30% (w/v) linear sucrose gradient. The ribosomal complexes were sedimented by ultracentrifugation for 3 h at 4 °C and 30,000 rpm using a Beckman SW41 swing bucket rot" @default.
• W2144195875 created "2016-06-24" @default.
• W2144195875 creator A5055978396 @default.
• W2144195875 creator A5086243160 @default.
• W2144195875 creator A5088960519 @default.
• W2144195875 date "2004-07-01" @default.
• W2144195875 modified "2023-10-16" @default.
• W2144195875 title "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" @default.
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