HCV IRES翻译启动活性的细胞特异性及其机制研究
|
摘要
目的：HCV 5'端非编码区含内部核糖体进入位点(IRES),其功能主要是调控病毒基因复制和以非帽依赖方式启动翻译病毒蛋白,而且HCV 5'UTR在不同基因型和株中呈现高度的序列保守,这也使它成为了抗病毒药物研发的良好靶位。本实验旨在研究在不同宿主细胞的翻译体系下,缺失不同结构域的HCV 5'UTR翻译启动活性的差异,并探索其机制。
方法：(1)以脂质体介导基因转染技术,将HCV 5'UTR缺失不同结构域并调控Flue的真核表达质粒与调控Rluc的真核表达质粒pRL-TK共转染至不同的细胞中,转染后36h。①提取细胞RNA进行半定量RT-PCR检测,利用质粒pRL-TK校正其它质粒的转染效率；②用双荧光素酶报告基因检测系统检测Fluc基因相对表达活性,分析HCV 5'UTR缺失不同结构域后在不同翻译体系中翻译启动活性的差异。(2)选择同种细胞中活性差异较大的缺失HCV 5'UTR不同结构域的质粒进行体外转录,获得RNA,使之与细胞蛋白质进行特异性结合,以筛选出特异性的结合蛋白。
结果：(1)缺失HCV 5'UTR的DomainⅠ及下游的单链序列后,HCV IRES的活性分别与缺失前相比：在Hela细胞和C6细胞中无明显影响,在L-02细胞中活性下降为46%,而在293T细胞则为缺失前的146%。(2)缺失HCV 5'UTR的DomainⅡ后,HCV IRES的活性分别与缺失前相比：在Hela细胞中,缺失后活性仅为缺失前的49%,而在L-02细胞、C6细胞和293T细胞中,活性分别为缺失前的140%、160%和235%。(3)体外转录出了缺失HCV 5'UTR不同结构域的RNA,并分离出了能与其特异性结合的蛋白质。
结论：(1)HCV 5'UTR的DomainⅠ对HCV IRES在L-02细胞株中的翻译启动活性具促进作用,而在293T细胞株中有抑制作用,在C6细胞株和Hela细胞株中DomainⅠ对IRES翻译启动活性无明显作用。(2)HCV 5'UTR的DomainⅡ对HCV IRES在Hela细胞株中的翻译启动活性有促进作用,而对其他三种细胞株却有抑制作用。(3)利用链霉亲和素磁珠将体外转录出来的RNA与细胞蛋白结合,发现了特异性结合的蛋白质,但尚未发现差异蛋白。
Objective:HCV 5'UTR contains an internal ribosome entry site (IRES), and its main functions are to regulate viral replication and to translate viral protein in cap-independent manner. In addition, the nucleotide sequence of HCV 5'UTR is highly conserved in different genotypes and strains, which makes it a good target for antiviral-drug research. The aim of this study was to analysis the differences of translation initiation activity of HCV 5'UTR with different deletions in different host cell lines and meanwhile to explore the mechanism.
Methods:(1)Through the liposome-mediated gene-transfection technology, the Flue eukaryotic expression plasmid regulated by HCV IRES with different deletions and the Rluc eukaryotic expression plasmid into different cell lines. At 36 posttransfection:①Total cellular RNA were harvested and detected by semiquantitative RT-PCR, and the pRL-TK plasmid was co-transfected as a nomalization cotrol;②The Flue gene's relative expression activity was detected through dual luciferase reporter gene assay system, and the different translation initiation activity was analysed in the different translation systems with different deletions of HCV IRES. (2)The plasmids with different deletions, which had significant difference in translational initiational activity, were selected, and those RNAs were in vitro transcripted to isolate specifically binding proteins.
Results:(1)Between the activity of the full length HCV IRES and those with the deletion of the Domain I and the downstream single-stranded sequence, no significant difference was observed in Hela cells and C6 cells, however, the translational activity was decreased by 46% in L-02 cells and increased by 46% in 293T cells by the deletion. (2) Between the activity of the full length HCV IRES and those with the deletion of Domain II, in Hela cells, the activity was decreased to 51%, while in L-02 cells, C6 cells and 293T cells, it increased 40%,60% and 135% respectively. (3)The RNAs of HCV 5'UTR with different deletions were in vitro transcripted and the cellular proteins which can specifically bind to the RNAs were isolated.
Conclusions:①In L-02 cells, the Domain I of HCV 5'UTR could promote the initiation translation activity of HCV IRES, while it showed inhibitory effect in 293T cell lines and had no effect in C6 cells and Hela cells.②Domain II of HCV 5'UTR could enhance the translation initiation activity of HCV IRES in Hela cells, but inhibit it in the other cell lines.③Binding the in vitro transcribed RNA with the cellular protein by the streptavidin-biotin and magnetic beads, the specific binding protein were isolated. Still, no different proteins were observed among the different deletions of RNAs.
方法：(1)以脂质体介导基因转染技术,将HCV 5'UTR缺失不同结构域并调控Flue的真核表达质粒与调控Rluc的真核表达质粒pRL-TK共转染至不同的细胞中,转染后36h。①提取细胞RNA进行半定量RT-PCR检测,利用质粒pRL-TK校正其它质粒的转染效率；②用双荧光素酶报告基因检测系统检测Fluc基因相对表达活性,分析HCV 5'UTR缺失不同结构域后在不同翻译体系中翻译启动活性的差异。(2)选择同种细胞中活性差异较大的缺失HCV 5'UTR不同结构域的质粒进行体外转录,获得RNA,使之与细胞蛋白质进行特异性结合,以筛选出特异性的结合蛋白。
结果：(1)缺失HCV 5'UTR的DomainⅠ及下游的单链序列后,HCV IRES的活性分别与缺失前相比：在Hela细胞和C6细胞中无明显影响,在L-02细胞中活性下降为46%,而在293T细胞则为缺失前的146%。(2)缺失HCV 5'UTR的DomainⅡ后,HCV IRES的活性分别与缺失前相比：在Hela细胞中,缺失后活性仅为缺失前的49%,而在L-02细胞、C6细胞和293T细胞中,活性分别为缺失前的140%、160%和235%。(3)体外转录出了缺失HCV 5'UTR不同结构域的RNA,并分离出了能与其特异性结合的蛋白质。
结论：(1)HCV 5'UTR的DomainⅠ对HCV IRES在L-02细胞株中的翻译启动活性具促进作用,而在293T细胞株中有抑制作用,在C6细胞株和Hela细胞株中DomainⅠ对IRES翻译启动活性无明显作用。(2)HCV 5'UTR的DomainⅡ对HCV IRES在Hela细胞株中的翻译启动活性有促进作用,而对其他三种细胞株却有抑制作用。(3)利用链霉亲和素磁珠将体外转录出来的RNA与细胞蛋白结合,发现了特异性结合的蛋白质,但尚未发现差异蛋白。
Objective:HCV 5'UTR contains an internal ribosome entry site (IRES), and its main functions are to regulate viral replication and to translate viral protein in cap-independent manner. In addition, the nucleotide sequence of HCV 5'UTR is highly conserved in different genotypes and strains, which makes it a good target for antiviral-drug research. The aim of this study was to analysis the differences of translation initiation activity of HCV 5'UTR with different deletions in different host cell lines and meanwhile to explore the mechanism.
Methods:(1)Through the liposome-mediated gene-transfection technology, the Flue eukaryotic expression plasmid regulated by HCV IRES with different deletions and the Rluc eukaryotic expression plasmid into different cell lines. At 36 posttransfection:①Total cellular RNA were harvested and detected by semiquantitative RT-PCR, and the pRL-TK plasmid was co-transfected as a nomalization cotrol;②The Flue gene's relative expression activity was detected through dual luciferase reporter gene assay system, and the different translation initiation activity was analysed in the different translation systems with different deletions of HCV IRES. (2)The plasmids with different deletions, which had significant difference in translational initiational activity, were selected, and those RNAs were in vitro transcripted to isolate specifically binding proteins.
Results:(1)Between the activity of the full length HCV IRES and those with the deletion of the Domain I and the downstream single-stranded sequence, no significant difference was observed in Hela cells and C6 cells, however, the translational activity was decreased by 46% in L-02 cells and increased by 46% in 293T cells by the deletion. (2) Between the activity of the full length HCV IRES and those with the deletion of Domain II, in Hela cells, the activity was decreased to 51%, while in L-02 cells, C6 cells and 293T cells, it increased 40%,60% and 135% respectively. (3)The RNAs of HCV 5'UTR with different deletions were in vitro transcripted and the cellular proteins which can specifically bind to the RNAs were isolated.
Conclusions:①In L-02 cells, the Domain I of HCV 5'UTR could promote the initiation translation activity of HCV IRES, while it showed inhibitory effect in 293T cell lines and had no effect in C6 cells and Hela cells.②Domain II of HCV 5'UTR could enhance the translation initiation activity of HCV IRES in Hela cells, but inhibit it in the other cell lines.③Binding the in vitro transcribed RNA with the cellular protein by the streptavidin-biotin and magnetic beads, the specific binding protein were isolated. Still, no different proteins were observed among the different deletions of RNAs.
引文
[1]Lindenbach BD, Rice CM. Unravelling hepatitis C virus replication from genome to function[J]. Nature,2005,436:933-938.
[2]梁雪松,安艳.针对宿主细胞靶位抗HCV策略研究进展[J].世界华人消化杂志,2007,15(15)：1754-1758.
[3]Poynard T, Ratziu V, Benhamou Y, et al. Natural history of HCV infection [J]. Bailliere's Clinical Gastroenterology2000,14(2):211-228.
[4]凌世淦.丙型肝炎疫苗研究的挑战与希望[J].中华微生物学和免疫学杂志2004,24(11)：921-922.
[5]Honda M, Beard MR, Ping LH, et al. A phylogenetically conserved stem-loop structure at the 5'border ofthe internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation[J]. J Virol,1999,73(2):1165-1174.
[6]Ji H, Fraser CS, Yu Y, et al. Coordinated assembly of human translationinitiation complexes by the hepatitis C virus internal ribosome entry site RNA[J]. Proc Natl Acad Sci U S A,2004,101(49):16990-16995.
[7]Otto GA, Puglisi JD. The pathway of HCV IRES-mediatcd translation initiation[J]. Cell,2004,119(3):369-380.
[8]Tsukiyama-Kohara K, Iizuka N, Kohara M, et al. Internal ribosome entry site within hepatitis C virus RNA[J]. J Virol,1992,66:1476-1483.
[9]Wang C, Sarnow P, Siddiqui A. Translation if human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism[J]. J Virol, 1993,67:3338-3344.
[10]Reynold JE, Kaminski A, Kettinen HJ, et al. Unique features of internal initiation of hepatitis C virus RN A translation[J]. J EMBO,1995,14:6010-6020.
[11]Honda M, Ping LH, Rijinbrand RC, Amphlett E. Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA[J]. Virology,1996,222 (1):31-42.
[12]Domitrovicha AM, Diebela KW, Ali N, et al. Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication[J]. Virology,2005,335: 72-86.
[13]Takayoshi S, Masao H, Hideki M, et al. La protein for internal ribosome enty site-directed translation is a protential therapeutic target for hepatitis C virus replication[J]. Journal of Infectious Diseases,2010,202 (1):75-85.
[14]Spangberg K, Goobar-Larsson L, Wahren-Herlenius M, et al. The La protein from human liver cells interacts specifically with the U-rich region in the hepatitis C virus 3 untranslated region[J]. J Hum Virol,1999,2:296-307.
[15]Aizaki H, Choi KS, Liu M, et al. Polypyrimidine-tract-binding protein is a component of the HCV RNAreplication complex and necessary for RNA synthesis[J]. Journal of Biomedical Science,2006,13:469-480.
[16]Ali N, Siddiqui A. Interaction of polypurimidine tract-binding protein with the 5 noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation[J]. J Virol,1995,69 (10): 6367-6375.
[17]Spangberg K, Schwartz S. Poly(C)-binding protein interacts with the hepatitis C virus 5'untranslated region[J]. J Gen Virol,1999,80:1371-1376.
[18]Dori M, Landry, Marala I, Hertz, et al. RPS25 isessential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs[J]. Gene Development,2009, 23:2753-2764.
[19]Paek KY, Kim CS, Park SM, et al. RNA-binding protein hnRNP D modulates internal ribosome entry site-dependent translation of hepatitis C virus RNA[J]. J Virol,2008,82 (24):12082-12093.
[20]Hwang B, Lim JH, Hahm B, Jang SK. hnRNP is required for the translation mediated by HCV IRES[J]. Biochem Biophys Res Commun,2009,378 (3): 584-588.
[21]Liu HM, Aizaki H, Choi KS, et al. SYNCRIP(synaptotagmin-binding, cytoplasmic RNA-interacting protein)is a host factor involved in hepatitis C virus RNA replication[J]. Virologu,2009,386 (2):249-256.
[22]Kim JH, Paek KY, Ha SH, et al. A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon[J]. Molecular and Cellular Biology, 2004,24(18):7878-7890.
[23]Choi KS, Mizutani A, Michael MC. SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis[J]. J Virol,2004,78 (23):13153-13162.
[24]Scheller N, Mina LB, Galao RP, et al. Tranlation and replication of hepatitis C virus genpmic RNA depends on ancient cellular proteins that control mRNA fates[J]. Proc Natl Acad Sci USA,2009,106 (32):13517-13522.
[25]Laporte J, Malet I, Andrieu T, et al. Comparative analusis of translation efficiencies of hepatitis C virus 5'untranslated regions among intraindividual quasispecies present in chronic infection:opposite behaviors depending on cell type[J]. J Virol, 2000,74 (22):10827-10833.
[26]Laporte J, Bain C, Maure P, et al. Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells[J]. Blood,2003,101 (1):52-57.
[27]Kamoshita N, Tsuldyama-Kohara K, Kohara M, et al. Genetic analysis ofinternal ribosomal entry site on hepatitis C virus RNA:implication forinvolvement of the highly ordered structure and cell type-specific transacting factors[J]. Virology,1997, 233(1):9-18.
[28]Lancaster AM, Jan E, Samow P. Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site[J]. RNA,2006,12(5):894-902.
[29]Resenfeld AB, Racaniello VR. Hepatitis C virus internal ribosome entry site-dependent translation in saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein 2, and La protein[J]. J Virol,2005,79(16): 10126-10137.
[30]Wakita T, Wands JR. Specific inhibition of hepatitis C virus expression byantisense oligodcoxynuclcetides. In vitro model for seleedtion of target sequence[J]. J Biol Chem,1994,269(19):14205-14210.
[31]李勇年,王勤环,斯崇文,等.丙型肝炎病毒基因调控细胞模型的建立及其意义[J].中华医学杂志,1997,77(8)：64-70.
[32]Alt M, Renz R, Hofschneider PH, et al. Specific inhibition of hepatitis C viralgene expression by antisense phosphorothinate oligodcoxynucleotides[J]. Hepatology, 1995,22(3):707-717.
[33]王小红,王升启,朱宝珍.反义寡核苷酸对HCV5'NCR基因调控功能的特异性抑制作用[J].中华微生物学和免疫学杂志,2000：20(5)：436-437.
[34]Rijnbrand R, Bredenbeek P, van der Straaten T, et al. Almost the entire 5'non-translated region of hepatitis C virus is required for cap-independent translation[J]. FEBS Letters,1995,365:115-119.
[35]Wang C, Sarnow P, Siddiqui A. A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA. J Virol,1994,68(11):7301-7307.
[36]刘水平,赵俊琴,谭德明,等.丙型肝炎病毒5'端非编码区的5端序列对其翻译启动活性的影响.胃肠病学和肝病学杂志,2005,14(4)：355-358.
[37]Yoo BJ, Spaete RR, Geballe AP, et al.5'end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5'untranslated region. Virology,1992, 191:889-899.
[38]Fukushi S, Kurihara C, lshiyama N, et al. The sequence element of the internal ribosome entry site and a 25-kilodahon cellular protein contribute to efficient internal initiation of translmion of hepatitis C virus RNA. J Virol,1997,71(2): 1662-1666.
[39]Dumas E, Staedel C, Colombat M, et al. A promoter activity is present in the DNA sequence corresponding tO the hepatitis C virus5'UTR [J]. Nucleic Acids Res.2003,31(4):1275-1281.
[40]Pestova TV, Shatsky IN, Fletcher SP, et al. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev,1998, 12(1):67-83.
[41]Spahn CM, Kieft JS, Grassucci RA, et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science,2001,291(5510): 1959-1962.
[42]张景霞,苏海霞,李端,等.紫外交联实验筛选HCV RNA结合蛋白的应用.中国公共卫生,2006,22(3)：289-290.
[43]孔令青,李勇,高洪,严玉霖,等.生物素-亲和素标记技术[J].动物医学进展,2008,29(4)：100-102.
[44]刘福英,宋淑霞,郑龙,等.链霉亲和素纯化和鉴定方法的研究[J].微生物学通报,2006,33(5)：112-116.
[45]裴仁军,崔小强,杨秀荣,等.实时生物分子相互作用分析技术用于链霉亲和素-生物素化抗体的层层组装研究[J].高等学校化学学报,2002,23(2)：195-198.
[46]吴在荣,王宏锐.链霉亲和素-生物素化学发光免疫分析血清胰岛素方法建立及临床应用[J].放射免疫学杂志,2010,23(5)：560-563.
[47]Tischendorf JJW, Beger C, Korf M, et al. Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication[J]. Arch Virol,2004,149 (10): 1955-1970.
[1]梁雪松,安艳.针对宿主细胞靶位抗HCV策略研究进展[J].世界华人消化杂志,2007,15(15)：1754—1758.
[2]Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture[J].Science,2000,290:1972-1974.
[3]Stephanie TS, Ki-Jeong L, Aizaki H, et al. Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2[J]. J Virol,2003, 7 (77):4160-4168.
[4]Lai MM. Cellular factors in the transcription and replication of viral RNA genomes:a parallel to DNAdependent RNA transcription[J]. J Virol,1998,244:1-12.
[5]McBride A E, A Schlegel, K Kirkegaard. Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells[J]. Proc. Natl. Acad. Sci., 1996,93:2296-2301.
[6]Tang H, Gaietta GM, Fischer WH, et al. A cellular cofactor for the constitutive transport element of type D retrovirus[J]. Science,1997,276:1412-1415.
[7]Agis-Juarez, Raul Azae, Galvan, et al. Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells[J]. J Gen Virol,2009,90 (Pt12):2893-2901.
[8]Angela M, Domitrovicha, Kevin W, et al. Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication[J]. Virology,2005,335:72-86.
[9]Ghetti A, Pinol-Roma S, Michael WM, et al. hnRNP I, the polypyrimidine tract-binding protein:distinct nuclear localization and association with hnRNAs Virology[J]. Nucleic Acids Res,1992,20:3671-3678.
[10]Kyung-Soo Chang, Guangxiang Luo. The polypyrimidine tract-binding protein (PTB) is required for efficient replication of hepatitis C virus (HCV) RNA[J]. Virus Research,2006,115:1-8.
[11]Hideki Aizaki, Keum SC, Minyi Liu, et al. Polyp yrimidine-tract-binding protein is a component of the HCV RNAreplication complex and necessary for RNA synthesis[J]. Journal of Biomedical Science,2006,13:469-480.
[12]Kim JH, Paek KY, Ha SH, et al. A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon[J]. Molecular and Cellular Biology, 2004,24(18):7878-7890.
[13]Choi KS, Mizutani A, Michael MC. SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis[J]. J Virol,2004,78 (23):13153-13162.
[14]Liu HM, Aizaki H, Choi KS, et al. SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a host factor involved in hepatitis C virus RNA replication[J]. Virology,2009,386:249-256.
[15]孙静慧,刘皋林,谭龙益,等.La蛋白与HBV RNA相互作用关系的研究进展[J].第二军医大学报,2004,25(9)：1020-1022.
[16]王春花,成军,郎振为,等.人自身La抗原蛋白与肝炎病毒的关系[J].世界华人消化杂志,2003,11(12)：1973—1976.
[17]Spangberg K, Goobar-Larsson L, Wahren-Herlenius M, Schwartz S. The La protein from human liver cells interacts specifically with the U-rich region in the hepatitis C virus 3 untranslated region[J]. J Hum Virol,1999,2:296-307.
[18]Gao L, Aizaki H, He JW, et al. Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft[J]. J Virol,2004,78 (7):3480-3488.
[19]Hamamoto I, Nishimura Y, Okamoto T, et al. Human VAP-B Is Involved in Hepatitis C Virus Replication through Interaction with NS5A and NS5B[J]. J Virol,2005,79 (21):13473-13482.
[20]Goh PY, Tan YJ, Lim SP, et al. Cellular RNA helicase p68 relocalization and interaction with the hepatitis C virus (HCV) NS5B protein and the potential role of p68 in HCV RNA replication[J]. J Virol,2004,78:5288-5298.
[2]梁雪松,安艳.针对宿主细胞靶位抗HCV策略研究进展[J].世界华人消化杂志,2007,15(15)：1754-1758.
[3]Poynard T, Ratziu V, Benhamou Y, et al. Natural history of HCV infection [J]. Bailliere's Clinical Gastroenterology2000,14(2):211-228.
[4]凌世淦.丙型肝炎疫苗研究的挑战与希望[J].中华微生物学和免疫学杂志2004,24(11)：921-922.
[5]Honda M, Beard MR, Ping LH, et al. A phylogenetically conserved stem-loop structure at the 5'border ofthe internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation[J]. J Virol,1999,73(2):1165-1174.
[6]Ji H, Fraser CS, Yu Y, et al. Coordinated assembly of human translationinitiation complexes by the hepatitis C virus internal ribosome entry site RNA[J]. Proc Natl Acad Sci U S A,2004,101(49):16990-16995.
[7]Otto GA, Puglisi JD. The pathway of HCV IRES-mediatcd translation initiation[J]. Cell,2004,119(3):369-380.
[8]Tsukiyama-Kohara K, Iizuka N, Kohara M, et al. Internal ribosome entry site within hepatitis C virus RNA[J]. J Virol,1992,66:1476-1483.
[9]Wang C, Sarnow P, Siddiqui A. Translation if human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism[J]. J Virol, 1993,67:3338-3344.
[10]Reynold JE, Kaminski A, Kettinen HJ, et al. Unique features of internal initiation of hepatitis C virus RN A translation[J]. J EMBO,1995,14:6010-6020.
[11]Honda M, Ping LH, Rijinbrand RC, Amphlett E. Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA[J]. Virology,1996,222 (1):31-42.
[12]Domitrovicha AM, Diebela KW, Ali N, et al. Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication[J]. Virology,2005,335: 72-86.
[13]Takayoshi S, Masao H, Hideki M, et al. La protein for internal ribosome enty site-directed translation is a protential therapeutic target for hepatitis C virus replication[J]. Journal of Infectious Diseases,2010,202 (1):75-85.
[14]Spangberg K, Goobar-Larsson L, Wahren-Herlenius M, et al. The La protein from human liver cells interacts specifically with the U-rich region in the hepatitis C virus 3 untranslated region[J]. J Hum Virol,1999,2:296-307.
[15]Aizaki H, Choi KS, Liu M, et al. Polypyrimidine-tract-binding protein is a component of the HCV RNAreplication complex and necessary for RNA synthesis[J]. Journal of Biomedical Science,2006,13:469-480.
[16]Ali N, Siddiqui A. Interaction of polypurimidine tract-binding protein with the 5 noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation[J]. J Virol,1995,69 (10): 6367-6375.
[17]Spangberg K, Schwartz S. Poly(C)-binding protein interacts with the hepatitis C virus 5'untranslated region[J]. J Gen Virol,1999,80:1371-1376.
[18]Dori M, Landry, Marala I, Hertz, et al. RPS25 isessential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs[J]. Gene Development,2009, 23:2753-2764.
[19]Paek KY, Kim CS, Park SM, et al. RNA-binding protein hnRNP D modulates internal ribosome entry site-dependent translation of hepatitis C virus RNA[J]. J Virol,2008,82 (24):12082-12093.
[20]Hwang B, Lim JH, Hahm B, Jang SK. hnRNP is required for the translation mediated by HCV IRES[J]. Biochem Biophys Res Commun,2009,378 (3): 584-588.
[21]Liu HM, Aizaki H, Choi KS, et al. SYNCRIP(synaptotagmin-binding, cytoplasmic RNA-interacting protein)is a host factor involved in hepatitis C virus RNA replication[J]. Virologu,2009,386 (2):249-256.
[22]Kim JH, Paek KY, Ha SH, et al. A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon[J]. Molecular and Cellular Biology, 2004,24(18):7878-7890.
[23]Choi KS, Mizutani A, Michael MC. SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis[J]. J Virol,2004,78 (23):13153-13162.
[24]Scheller N, Mina LB, Galao RP, et al. Tranlation and replication of hepatitis C virus genpmic RNA depends on ancient cellular proteins that control mRNA fates[J]. Proc Natl Acad Sci USA,2009,106 (32):13517-13522.
[25]Laporte J, Malet I, Andrieu T, et al. Comparative analusis of translation efficiencies of hepatitis C virus 5'untranslated regions among intraindividual quasispecies present in chronic infection:opposite behaviors depending on cell type[J]. J Virol, 2000,74 (22):10827-10833.
[26]Laporte J, Bain C, Maure P, et al. Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells[J]. Blood,2003,101 (1):52-57.
[27]Kamoshita N, Tsuldyama-Kohara K, Kohara M, et al. Genetic analysis ofinternal ribosomal entry site on hepatitis C virus RNA:implication forinvolvement of the highly ordered structure and cell type-specific transacting factors[J]. Virology,1997, 233(1):9-18.
[28]Lancaster AM, Jan E, Samow P. Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site[J]. RNA,2006,12(5):894-902.
[29]Resenfeld AB, Racaniello VR. Hepatitis C virus internal ribosome entry site-dependent translation in saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein 2, and La protein[J]. J Virol,2005,79(16): 10126-10137.
[30]Wakita T, Wands JR. Specific inhibition of hepatitis C virus expression byantisense oligodcoxynuclcetides. In vitro model for seleedtion of target sequence[J]. J Biol Chem,1994,269(19):14205-14210.
[31]李勇年,王勤环,斯崇文,等.丙型肝炎病毒基因调控细胞模型的建立及其意义[J].中华医学杂志,1997,77(8)：64-70.
[32]Alt M, Renz R, Hofschneider PH, et al. Specific inhibition of hepatitis C viralgene expression by antisense phosphorothinate oligodcoxynucleotides[J]. Hepatology, 1995,22(3):707-717.
[33]王小红,王升启,朱宝珍.反义寡核苷酸对HCV5'NCR基因调控功能的特异性抑制作用[J].中华微生物学和免疫学杂志,2000：20(5)：436-437.
[34]Rijnbrand R, Bredenbeek P, van der Straaten T, et al. Almost the entire 5'non-translated region of hepatitis C virus is required for cap-independent translation[J]. FEBS Letters,1995,365:115-119.
[35]Wang C, Sarnow P, Siddiqui A. A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA. J Virol,1994,68(11):7301-7307.
[36]刘水平,赵俊琴,谭德明,等.丙型肝炎病毒5'端非编码区的5端序列对其翻译启动活性的影响.胃肠病学和肝病学杂志,2005,14(4)：355-358.
[37]Yoo BJ, Spaete RR, Geballe AP, et al.5'end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5'untranslated region. Virology,1992, 191:889-899.
[38]Fukushi S, Kurihara C, lshiyama N, et al. The sequence element of the internal ribosome entry site and a 25-kilodahon cellular protein contribute to efficient internal initiation of translmion of hepatitis C virus RNA. J Virol,1997,71(2): 1662-1666.
[39]Dumas E, Staedel C, Colombat M, et al. A promoter activity is present in the DNA sequence corresponding tO the hepatitis C virus5'UTR [J]. Nucleic Acids Res.2003,31(4):1275-1281.
[40]Pestova TV, Shatsky IN, Fletcher SP, et al. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev,1998, 12(1):67-83.
[41]Spahn CM, Kieft JS, Grassucci RA, et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science,2001,291(5510): 1959-1962.
[42]张景霞,苏海霞,李端,等.紫外交联实验筛选HCV RNA结合蛋白的应用.中国公共卫生,2006,22(3)：289-290.
[43]孔令青,李勇,高洪,严玉霖,等.生物素-亲和素标记技术[J].动物医学进展,2008,29(4)：100-102.
[44]刘福英,宋淑霞,郑龙,等.链霉亲和素纯化和鉴定方法的研究[J].微生物学通报,2006,33(5)：112-116.
[45]裴仁军,崔小强,杨秀荣,等.实时生物分子相互作用分析技术用于链霉亲和素-生物素化抗体的层层组装研究[J].高等学校化学学报,2002,23(2)：195-198.
[46]吴在荣,王宏锐.链霉亲和素-生物素化学发光免疫分析血清胰岛素方法建立及临床应用[J].放射免疫学杂志,2010,23(5)：560-563.
[47]Tischendorf JJW, Beger C, Korf M, et al. Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication[J]. Arch Virol,2004,149 (10): 1955-1970.
[1]梁雪松,安艳.针对宿主细胞靶位抗HCV策略研究进展[J].世界华人消化杂志,2007,15(15)：1754—1758.
[2]Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture[J].Science,2000,290:1972-1974.
[3]Stephanie TS, Ki-Jeong L, Aizaki H, et al. Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2[J]. J Virol,2003, 7 (77):4160-4168.
[4]Lai MM. Cellular factors in the transcription and replication of viral RNA genomes:a parallel to DNAdependent RNA transcription[J]. J Virol,1998,244:1-12.
[5]McBride A E, A Schlegel, K Kirkegaard. Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells[J]. Proc. Natl. Acad. Sci., 1996,93:2296-2301.
[6]Tang H, Gaietta GM, Fischer WH, et al. A cellular cofactor for the constitutive transport element of type D retrovirus[J]. Science,1997,276:1412-1415.
[7]Agis-Juarez, Raul Azae, Galvan, et al. Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells[J]. J Gen Virol,2009,90 (Pt12):2893-2901.
[8]Angela M, Domitrovicha, Kevin W, et al. Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication[J]. Virology,2005,335:72-86.
[9]Ghetti A, Pinol-Roma S, Michael WM, et al. hnRNP I, the polypyrimidine tract-binding protein:distinct nuclear localization and association with hnRNAs Virology[J]. Nucleic Acids Res,1992,20:3671-3678.
[10]Kyung-Soo Chang, Guangxiang Luo. The polypyrimidine tract-binding protein (PTB) is required for efficient replication of hepatitis C virus (HCV) RNA[J]. Virus Research,2006,115:1-8.
[11]Hideki Aizaki, Keum SC, Minyi Liu, et al. Polyp yrimidine-tract-binding protein is a component of the HCV RNAreplication complex and necessary for RNA synthesis[J]. Journal of Biomedical Science,2006,13:469-480.
[12]Kim JH, Paek KY, Ha SH, et al. A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon[J]. Molecular and Cellular Biology, 2004,24(18):7878-7890.
[13]Choi KS, Mizutani A, Michael MC. SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis[J]. J Virol,2004,78 (23):13153-13162.
[14]Liu HM, Aizaki H, Choi KS, et al. SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a host factor involved in hepatitis C virus RNA replication[J]. Virology,2009,386:249-256.
[15]孙静慧,刘皋林,谭龙益,等.La蛋白与HBV RNA相互作用关系的研究进展[J].第二军医大学报,2004,25(9)：1020-1022.
[16]王春花,成军,郎振为,等.人自身La抗原蛋白与肝炎病毒的关系[J].世界华人消化杂志,2003,11(12)：1973—1976.
[17]Spangberg K, Goobar-Larsson L, Wahren-Herlenius M, Schwartz S. The La protein from human liver cells interacts specifically with the U-rich region in the hepatitis C virus 3 untranslated region[J]. J Hum Virol,1999,2:296-307.
[18]Gao L, Aizaki H, He JW, et al. Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft[J]. J Virol,2004,78 (7):3480-3488.
[19]Hamamoto I, Nishimura Y, Okamoto T, et al. Human VAP-B Is Involved in Hepatitis C Virus Replication through Interaction with NS5A and NS5B[J]. J Virol,2005,79 (21):13473-13482.
[20]Goh PY, Tan YJ, Lim SP, et al. Cellular RNA helicase p68 relocalization and interaction with the hepatitis C virus (HCV) NS5B protein and the potential role of p68 in HCV RNA replication[J]. J Virol,2004,78:5288-5298.