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1 Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan, ROC
2 Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, Taiwan, ROC
3 Graduate Program in Biomedical Science, Chang Gung University, Taoyuan, Taiwan, ROC
4 Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA
Correspondence
Shin-Ru Shih
srshih{at}mail.cgu.edu.tw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; McMinn, 2002; McMinn et al., 2001). In recent years, an increase in EV71 epidemic activity has been noted throughout the Asia–Pacific region. Severe neurological complications, including brainstem encephalitis, meningitis, poliomyelitis and even death have occurred in these epidemics (McMinn, 2002). In 1998, an EV71 epidemic occurred in Taiwan, with the virus infecting over 120 000 people and killing 78 children (Ho et al., 1999). Many EV71 epidemics on a smaller scale also occurred after 1998 on the island (Chen et al., 2007).
EV71, a member of the family Picornaviridae, is a positive-stranded RNA virus (Brown & Pallansch, 1995). The viral RNA has a small protein called VPg covalently attached to its 5' end and is polyadenylated at its 3' terminus (Flanegan et al., 1977; Sarnow, 1989). The genomic RNA is around 7500 nt. The 5' untranslated region (UTR) is 745 nt and is highly structured, containing a cloverleaf-like structure important for viral RNA synthesis and an internal ribosomal entry site (IRES) that is critical for the direction of viral mRNA translation (Thompson & Sarnow, 2003). Many cellular proteins interact with the picornavirus 5' UTR and regulate virus replication; for instance, poly(rC)-binding protein (PCBP), an RNA-binding protein that contains three heterogeneous nuclear ribonucleoprotein K (hnRNP K) homology (KH) motifs, has been demonstrated to interact with the 5' UTR of poliovirus and rhinovirus (Walter et al., 1999, 2002). Various IRES-specific trans-acting factors, such as polypyrimidine tract-binding protein (PTB), PCBP, autoantigen La and upstream N-ras protein (Unr), have been reported to be functionally important for picornaviruses (Boussadia et al., 2003; Costa-Mattioli et al., 2004; Toyoda et al., 1994; Walter et al., 1999).
Knowledge of cellular proteins that associate with the 5' UTR of picornaviruses would facilitate an understanding of virus–host interactions that are crucial molecular targets for antiviral drug development. In this study, streptavidin was used to pull down biotin-labelled EV71 5' UTR and its associated cellular proteins. Fourteen bands representing 11 proteins were identified as potential EV71 5' UTR-associated proteins. hnRNP K was one such protein and was chosen for further study. hnRNP K has been reported to be involved in many viral infections (Bryant et al., 2000; Burnham et al., 2007; Chang et al., 2001; Hsieh et al., 1998; Shimada et al., 2004; Zhang et al., 2008), but its role in picornavirus infection remains unclear. The interaction domains in both the viral RNA and the hnRNP K protein were mapped. The impact of hnRNP K on EV71 virus replication was also addressed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of proteins associated with EV71 5' UTR RNA sequences by affinity binding to biotinylated RNA.
A reaction mixture containing 200 µg cell extract and 12.5 pmol biotinylated EV71 5' UTR RNA probe was prepared. The mixture (with a final volume of 100 µl) contained 5 mM HEPES (pH 7.1), 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 2 mM dithiothreitol, 1 U RNasin and 0.25 mg heparin ml–1 (RNA mobility shift buffer) and was incubated for 15 min at 30 °C and then added to 400 µl Streptavidin MagneSphere Paramagnetic Particles (Promega) for 10 min at room temperature to allow binding. The protein–RNA complexes were washed five times with the RNA mobility shift buffer without heparin. After the final wash, 30 µl 2x SDS-PAGE sample buffer was added to the beads and incubated for 10 min at room temperature to dissociate the proteins from the RNA. The sample containing the eluted proteins was then boiled and subjected to 8–16 % SDS-PAGE and visualized by silver staining or Western blotting. Proteins bands were excised and identified by in-gel trypsin digestion and analysed by Bruker Ultraflex matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Database-searching algorithm.
After the masses derived from the standards, trypsin, the matrix and keratins had been removed, the monoisotopic mass lists for each protonated peptide were used to search the database. Mass lists were exported to the Biotool 2.0 software package to perform peptide mass fingerprinting using Mascot (http://www.matrixscience.com) algorithm scoring to identify the proteins.
Western blot analysis.
PVDF membranes were blocked and probed with antibodies against hnRNP K (diluted 1 : 200; Santa Cruz Biotechnology), IGF-II mRNA-binding protein (IMP-1; diluted 1 : 500; Santa Cruz Biotechnology) and actin (diluted 1 : 5000; Chemicon).
Co-immunoprecipitation.
Cell extracts from EV71-infected RD cells for use in co-immunoprecipitation assays were prepared at 6 h post-infection (p.i.). Lysates were pre-cleared by incubation on ice for 1 h with protein A–agarose (50 % in lysis buffer) bound to non-specific antibody. Non-specific complexes were pelleted by centrifugation at 10 000 g at 4 °C for 10 min. The supernatants were removed and used in the immunoprecipitation assay. Next, 100 µl pre-cleared lysate was diluted with 450 µl lysis buffer and then added to 15 µl hnRNP K antibody, followed by incubation on ice for 2 h. Pre-washed protein A–agarose (100 µl in PBS; 50 : 50) was added to each sample, which was then incubated on ice for 1 h. Immune complexes were pelleted by centrifugation at 1000 g at 4 °C for 5 min and washed three times with lysis buffer. Each pellet [or 100 µl pre-cleared lysate (total RNA)] was resuspended in 400 µl proteinase K buffer [100 mM Tris/HCl (pH 7.5), 12.5 mM EDTA, 150 mM NaCl, 1 % SDS] and incubated with 100 µg pre-digested proteinase K for 30 min at 37 °C. RNA was extracted with phenol/chloroform, precipitated in ethanol at –20 °C for 1 h, washed in 70 % ethanol, dried and resuspended in 20 µl DEPC H2O.
Fluorescence microscopy analysis.
RD cells grown on glass coverslips were infected with EV71 for 1 h at an m.o.i. of 40. At 6 h p.i., the culture medium was removed and the cells were washed and fixed. The cells were then permeabilized in 5 % Triton X-100 at room temperature for 5 min. For hnRNP K and EV71 3A immunostaining, the samples were blocked in PBS containing 5 % BSA for 60 min at room temperature and then incubated with anti-hnRNP K antibody (diluted 1 : 200) and anti-EV71 3A antibody (diluted 1 : 200) for 1.5 h at room temperature. The samples were then reacted with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After washing with PBS, the samples were treated with the nuclear stain Hoechst 33258 for 15 min and washed again three times with PBS. Images were captured using a confocal laser-scanning microscope (LSM 510 META; Zeiss).
Virus growth and plaque assay.
RD cells were transfected with hnRNP K and negative-control (NC) small interfering RNA (siRNA) for 48 h, trypsinzed and then counted. Cells (2x105) were seeded into 12-well plates. After 24 h, the cells were challenged with EV71 (strain 4643/TW/1998) at an m.o.i. of 40 or 0.1 p.f.u. per cell. At various times p.i., the supernatants of cell culture medium and cell lysates were collected to determine viral titres by plaque assay on RD cells. The sequences of siRNA used were as follows: hnRNP K siRNA: 5'-AAUUCCUCCUGCUAGACUCUGAUGA-3'; NC siRNA: 5'-AACUGGGUAAGCGGGCGCAAAUU-3'; IMP-1 siRNA: 5'-UACUGUACCAUACUGAGCCAGCAGG-3'.
Evaluation of RNA replication by slot blotting.
RD cells were transfected with hnRNP K and NC siRNA for 48 h and then trypsinzed and counted. The cells (2x105) were seeded into 12-well plates. After 24 h, the cells were challenged with EV71 (strain 4643/TW/1998) at an m.o.i. of 40 and harvested at 2, 3, 4, 5, 6, 7, 8 and 9 h p.i. RNA were extracted and dissolved in 20x SSC containing formaldehyde for 30 min at 60 °C. The reaction was then loaded onto a nitrocellulose membrane in the slot-blot manifold. After washing twice, the membrane was removed, air dried and cross-linked in a Stratalinker (Stratagene) at 200 J for 9 min. The membrane was pre-hybridized at 68 °C for 30 min in DIG Easy Hyb (Roche). Digoxygenin (DIG)-labelled RNA probes, specific for the genome or anti-genome, were produced using a DIG Northern Starter kit (Roche). After addition of the probes at 100 ng ml–1, the blots were incubated at 68 °C for 16 h. After the hybridization, the membrane was immediately submerged in a tray containing low-stringency buffer (2x SSC containing 0.1 % SDS) at room temperature for 5 min with shaking. The blot was then incubated twice (15 min each with shaking) in high-stringency buffer (0.1x SSC containing 0.1 % SDS) at 68 °C. The membrane was then incubated with Washing Buffer (Roche) for 2 min at room temperature with shaking. After the membrane had been blocked with Blocking Solution (Roche) for 30 min, it was incubated with alkaline phosphatase-conjugated anti-DIG antibody solution for 30 min and then washed twice with Maleic Acid Buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3 % Tween 20, pH 7.5; Roche). It was then equilibrated for 5 min in 20 ml Detection Buffer [0.1 M Tris/HCl (pH 9.5), 0.1 M NaCl; Roche]. Finally, chemiluminescent substrate (CDP-Star; Roche) was added and the membrane was exposed to Kodak film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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outlines the design of the pull-down assay. In comparison with the control reactions of no RNA, biotin-16-UTP only and unlabelled EV71 5' UTR (Fig. 1b, lanes 1–3), 14 bands of distinct sizes (Fig. 1b, lane 4) were observed in the mixture of biotinylated EV71 5' UTR RNA and RD cell lysate, which may represent cellular proteins that specifically associate with the EV71 5' UTR. These protein bands were excised, digested with trypsin and subjected to MALDI-TOF MS analysis. The results are shown in Table 1 with their accession numbers obtained from the NCBI protein database. They included four proteins known to interact with the IRES of picornaviruses: Unr (band 2), PTB-1 and PTB-2 (bands 8-1, 8-2, 9-1 and 9-2), PCBP2 (bands 11 and 12) and PCBP1 (band 13). Band 7 representing hnRNP K was chosen for further investigation in this study, as many studies have demonstrated that hnRNP K participates in viral infection (Bryant et al., 2000; Burnham et al., 2007; Chang et al., 2001; Hsieh et al., 1998; Shimada et al., 2004; Zhang et al., 2008), but the role of hnRNP K in picornavirus replication remains to be determined. The specific interaction between hnRNP K and the EV71 5' UTR was further verified by a competition assay and Western blotting. As shown in Fig. 1(c), this interaction was outcompeted by non-biotinylated EV71 5' UTR (Fig. 1c, lanes 3–5) but not by yeast tRNA (Fig. 1c, lanes 8–10) or EV71 VP1 RNA (Fig. 1c, lanes 13–15).
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(d, lanes 1–4). EV71 5' UTR RNA was pulled down from infected-cell lysate using anti-hnRNP K antibody. No specific band representing EV71 5' UTR was detected when the reaction proceeded without anti-hnRNP K antibody. As a negative control, primers specific to ribosomal protein S16 (RPS16) were used for RT-PCR (Fig. 1d, lanes 5–8).
hnRNP K localizes in the cytoplasm where EV71 replication occurs
hnRNP K is a nuclear protein, whereas EV71 replication occurs in the cytoplasm. EV71-infected cells were fixed at 6 h p.i. and stained with antibody against hnRNP K to determine its location. The results obtained by confocal microscopy clearly demonstrated that hnRNP K was localized in the nucleus of mock-infected cells (Fig. 2, upper panels); however, hnRNP K was enriched in the cytoplasm in EV71-infected cells (Fig. 2, lower panels). Staining with antibody against viral 3A protein was used to identify EV71-infected cells.
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shows the proposed secondary structure for the EV71 5' UTR as predicted by Mfold. Various truncated forms of viral RNA as indicated in Fig. 3(b) were synthesized by in vitro transcription and labelled using biotin. The pull-down assay was used as described above using streptavidin beads to capture the biotinylated viral RNA and associated cellular proteins from RD cells. Western blotting was then carried out to determine which regions of hnRNP K were interacting. Probes covering nt 1–167, 91–445, 91–561, 91–636, 91–745 and 242–445 (Fig. 3b, lanes 4, 8, 10, 12, 14 and 16, respectively) pulled down hnRNP K from RD cell lysate, whilst the other probes did not. These results suggest that hnRNP K may interact with the regions nt 1–167 (stem–loops I and II) and nt 242–445 (stem–loop IV) of the EV71 5' UTR.
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) were constructed. These plasmids were transfected individually into RD cells and the cell lysates were used in pull-down assays. Expression of full-length hnRNP K and its truncated forms was visualized by Western blotting using anti-Flag antibody (Fig. 4b, lanes 1, 4, 7, 10 and 13). Streptavidin was shown to capture the biotinylated EV71 5' UTR and its associated full-length hnRNP K (Fig. 4b, lane 3), as well as the two truncated forms of nt 1–385 and 144–465 (Fig. 4b, lanes 12 and 15), but not the forms of nt 1–92 and 1–197 (Fig. 4b, lanes 6 and 9). These results suggest that hnRNP K may interact with the EV71 5' UTR through a region containing KH2 and the proline-rich domain plus one neighbouring KH domain.
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, lower panels). As described in Methods, siRNA-transfected cells were collected, counted and reseeded into plates for further infection. RD cells that had been treated with NC siRNA or hnRNP K siRNA were then challenged with a high (m.o.i. of 40) or low (m.o.i. of 0.1) titre of EV71. Viral yields were measured at various times p.i. by plaque assay. Lower replication rates were observed in hnRNP K knockdown cells than in NC siRNA-treated cells (Fig. 5a), indicating that hnRNP K may play a positive role in EV71 replication. As a control, siRNA against IMP-1 (another cellular protein identified in Table 1) was used to treat the cells. The EV71 replication rate did not differ significantly in IMP-1 siRNA-transfected cells (Fig. 5b).
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(lower right panel) showed that hnRNP K was knocked down by its specific siRNA. EV71 (m.o.i. of 40) was used to infect cells and viral RNAs were extracted at different times p.i. A slot-blot assay using a specific RNA probe against either positive-sense or negative-sense EV71 viral RNA was employed to monitor viral RNA synthesis. The results in Fig. 6 demonstrated that the synthesis of both positive- and negative-strand EV71 viral RNA was delayed in hnRNP K knockdown cells. No significant difference in actin RNA amounts between NC and hnRNP K siRNA-treated cells was observed. These results suggest that hnRNP K may enhance viral RNA synthesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Parsley et al., 1997; Silvera et al., 1999). Another cellular protein critical for the synthesis of poliovirus RNA is poly(A)-binding protein (PABP). PABP interacts with PCBP, 3CD and the viral poly(A) tail to form a ribonucleoprotein complex that is required for negative-strand RNA synthesis (Herold & Andino, 2001; Wang et al., 1999). hnRNP C1 has also been found to interact with a secondary structure element that is predicted to form at the 3' end of the poliovirus negative-strand RNA and is functionally important for viral RNA synthesis (Brunner et al., 2005). In the present study of EV71 replication, another cellular protein, hnRNP K, was identified as interacting with the first 167 nt of the positive-stranded RNA genome. EV71 viral RNA synthesis was decreased when expression of the host cellular hnRNP K was shut down by siRNA. Whether hnRNP K interacts with other cloverleaf-associated proteins requires investigation to determine in detail the mechanism by which hnRNP K influences EV71 viral RNA synthesis.
In addition to the first 167 nt, hnRNP K also interacted with nt 242–445 of the EV71 5' UTR. Based on the RNA secondary structure predicted by Mfold, nt 242–445 may form stem–loop IV within the IRES, which is crucial for viral protein synthesis. hnRNP K participates in c-myc IRES activity (Evans et al., 2003), but has not been thought to be essential for poliovirus and rhinovirus viral protein translation (Choi et al., 2004). Our work did not clarify why hnRNP K interacts with the region nt 242–445. It may stabilize EV71 viral RNA through this interaction and thereby promote viral RNA synthesis.
hnRNP K is known to be a pre-mRNA-binding protein and shuttles intermediates between the nucleus and cytoplasm. It is involved in diverse molecular and cellular functions such as transcription, translation and nuclear–cytoplasmic shuttling (Bomsztyk et al., 2004). Our work revealed that hnRNP K localized mainly in the nucleus in mock-infected cells, whilst the protein was found mainly in the cytoplasm of EV71-infected cells at 6 h p.i. According to the results of a [35S]Met-labelling experiment (data not shown), viral protein synthesis dominated around this time point. Accordingly, hnRNP K becomes enriched in the cytoplasm when virus replication is proceeding. hnRNP K also reportedly changes its localization following Sindbis virus infection (Burnham et al., 2007). It is not clear how hnRNP K moves from the nucleus to the cytoplasm when the host cells are challenged by a virus. Identification of viral or host factors that participate in the mechanism of hnRNP K transportation would be of interest.
Recently, hnRNP K was also found to be important in regulating viral infection. For example, the overexpression of hnRNP K augmented hepatitis B virus (HBV) replication, whereas gene silencing of endogenous hnRNP K resulted in a significant reduction in the HBV viral load. Cytidine deaminase APOBEC3B interacts with hnRNP K and suppresses HBV expression (Zhang et al., 2008). hnRNP K has also been demonstrated to interact with Sindbis virus non-structural protein and viral subgenomic mRNA, and a reduction in hnRNP K expression in HeLa cells by siRNA treatment weakens expression of green fluorescent protein driven by a viral subgenomic promoter (Burnham et al., 2007). In addition to these examples, hnRNP K also interacts with several viral proteins, including human herpesvirus 6 immediate-early protein 2 (Shimada et al., 2004), dengue virus core protein (Chang et al., 2001), herpes simples virus type 1 IE63 protein (Bryant et al., 2000) and hepatitis C virus core protein (Hsieh et al., 1998).
The hnRNP K protein has three KH domains. A proline-rich domain is flanked by the KH2 and KH3 domains. KH stands for K homology, with reference to the initial identification of the motif in hnRNP K (Adinolfi et al., 1999; Messias & Sattler, 2004; Siomi et al., 1993; Tomonaga & Levens, 1995). The KH domain is a 45–55 aa motif that has been shown to have nucleic acid-binding activity. Numerous RNA-binding proteins contain a KH domain. However, they exhibit a diverse architecture (Dejgaard & Leffers, 1996; Gibson et al., 1993; Leopoldino et al., 2007; Siomi et al., 1993). Moreover, various KH domains bind to different nucleic acids. Here, we found that the interaction domain of hnRNP K with the EV71 5' UTR is located in the region containing KH2 and the proline-rich domain plus one neighbouring KH domain. The KH domain is responsible for RNA binding (Siomi et al., 1993), whilst the proline-rich domain is considered to be critical for the interaction of hnRNP K with other proteins. hnRNP K may bind to the EV71 5' UTR directly or through other viral and/or cellular proteins.
hnRNP K is not only important for virus replication; it has also frequently been reported to play a crucial role in cancer biology. A loss-of-function screening assay by randomized intracellular antibodies has demonstrated that hnRNP K is a potential target for metastasis (Inoue et al., 2007). Enhanced interaction between hnRNP K and nucleolin regulates gastrin mRNA turnover, which is related to gastrointestinal tract malignancies (Lee et al., 2007). hnRNP K drives translational activation of specifically silenced mRNAs, such as the L2 mRNA of human papillomavirus type 16 in squamous epithelial cells (Collier et al., 1998). These cited studies highlight the importance of hnRNP K in disease formation. We believe that further investigation of the details of the mechanism by which the hnRNP K switches among its multiple roles in various cells under different circumstances would be helpful in the development of therapeutic strategies against cancers and infectious diseases.
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ACKNOWLEDGEMENTS |
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Received 29 April 2008;
accepted 18 June 2008.
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  Y. Ma, J. Yu, H. L. Y. Chan, Y.-c. Chen, H. Wang, Y. Chen, C.-y. Chan, M. Y. Y. Go, S.-n. Tsai, S.-m. Ngai, et al. Glucose-regulated Protein 78 Is an Intracellular Antiviral Factor against Hepatitis B Virus Mol. Cell. Proteomics, November 1, 2009; 8(11): 2582 - 2594. [Abstract] [Full Text] [PDF]
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 T.-C. Chen, H.-Y. Chang, P.-F. Lin, J.-H. Chern, J. T.-A. Hsu, C.-Y. Chang, and S.-R. Shih Novel Antiviral Agent DTriP-22 Targets RNA-Dependent RNA Polymerase of Enterovirus 71 Antimicrob. Agents Chemother., July 1, 2009; 53(7): 2740 - 2747. [Abstract] [Full Text] [PDF]
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 J.-Y. Lin, S.-R. Shih, M. Pan, C. Li, C.-F. Lue, V. Stollar, and M.-L. Li hnRNP A1 Interacts with the 5' Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication J. Virol., June 15, 2009; 83(12): 6106 - 6114. [Abstract] [Full Text] [PDF]
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 J.-Y. Lin, M.-L. Li, and S.-R. Shih Far upstream element binding protein 2 interacts with enterovirus 71 internal ribosomal entry site and negatively regulates viral translation Nucleic Acids Res., January 1, 2009; 37(1): 47 - 59. [Abstract] [Full Text] [PDF]
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