丙型肝炎病毒结构区准种的优化筛选及其功能性蛋白的表达
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摘要
丙型肝炎病毒(HCV)是全球分布的重要病原体。急性HCV感染者约80％将转为持续性感染，且是引起肝硬化或肝癌的主要因素。HCV归属于黄病毒科(Flaviviridae family)的嗜肝病毒属，该病毒拥有约9.5 kb的正单链RNA基因组，含一个连续的开放读码框，可编码一种多聚前蛋白，由宿主和病毒的蛋白酶对此多聚前蛋白共同加工形成HCV病毒蛋白，包括位于N端的结构蛋白(structural protein)和位于C端的非结构蛋白(non-structural protein, NS)。HCV在复制过程中易发生变异，按其基因组序列异质性程度高低可分基因型(30％)、亚型(20％)及分离株(10％)。与其他RNA病毒一样，HCV最突出的变异，是多种相互密切关联而各不相同的病毒共存于同一宿主，称为准种(quasispecies)。HCV准种因能引起免疫逃避，导致HCV感染慢性化和影响干扰素治疗效果等，而成为当前的研究热点。
有关准种的研究近年来报道很多，但大多集中于HCV基因组变异最大的包膜2区(E2)N端的高度变异1区(HVR1)。研究表明，HVR1免疫逃逸株产生与病毒持续性感染有关。然而，研究证实，HCV异质性分布在整个基因组范围。持续性感染的HCV结构区可能还存在更多的有意义的变异区或位点。此外，准种研究的传统手段是分子克隆目标基因的PCR产物，并对每个克隆进行序列分析。要准确评估血标本中HCV准种的差异性，必须对大量克隆进行测序，但由于工作量大及开支高的原因，大多研究者仅对每例标本中的少量克隆进行测序。
本研究针对当前有关研究之不足，以急性HCV感染的两种转归即一过性及持续性感染者为对象，采用PCR扩增HCV结构区包括C区C端、
EI和EZ区N端长约 Ikb片段，并进行克隆，以单链构象多态性kingle strand
conformatlon polymorphism，SSCP）／异质性双体（heteroduplex，HD）分析为
准种筛选方法，分析了准种复杂性，并对所选的代表性准种进行核酸序列
分析及结构蛋白表达，以了解准种演变进化概貌及其与病情转归的关系。
1．HCV结构区基因的扩增及克隆
以5’NCR的限制性长度多态性分析为基因分型方法，采用混合碱基设
计的通用引物一次性扩增了 HCV la、fo及 3b型的 C肥用 区长约 Ikb片
断，以此提高准种检测的灵敏度。利用TA克隆载体PT－AdV对结构区基因
的P＊R产物进行克隆，使克隆效率＞90％。
2．HCV结构区准种的忧化筛选
选通过较长PCR扩增HCV结构区长约Ikb片断，继而分两组PCR
扩增 C／EI区 （47 hp）和 EZ／HVRI区 （57 hp），每仔病人随机选取一个克
隆的C／EI或EZ区扩增产物作为驱动子＊river卜 以SSCP用D方法对每份
标本的33个克隆的C用吧 区克隆型（准种）进行筛选。结果显示，每例
病人不同时相标本的EI 区克隆型存在廷续性；一过性感染者受感初期与
病毒清除前的EZ区优势克隆型虽不同，但未出现新的克隆型，而持续性感
染者急性期与漫性期血中的克隆型组成发生明显变化，并出现新的克隆型，
导致准种复杂性明显增加。
3．HCV准种的演变与急性感染的转归
对HCV一过性感染和持续性感染者不同时间标本的SSCP／HD优势克
隆型及劣势克隆型进行测序，通过序列差异性分析，观察HVRI及其他结
构区准种的演变状况；经第一时相对其他时相核酸序列的非同义碱基替换
和同义碱基替换率测定，进一步分析正性选择作用对准种演化的影响。结
果显示，持续性感染者中HVRI序列差异性明显增大，而一过性感染者则
无明显改变，两者的N及HVRI以外的EZ区序列差异性亦无明显差异，
表明一过性和持续性感染显示的准种演变形式不同主要由 HVR 变异所
致；持续性感染者HVRI的每月平均非同义碱基替换数呈增高趋势，两组
病人的m区和除外HVRI的EZ区＊02个氨基酸残基）平均非同义碱基
·IX·
、
替换数却保持低水平，相反，两组病人EI、HVRI及HVRI以外EZ区的
每月平均同义碱基替换数无显著差别，提示宿主兔疫压力主要对HVRI产
生阳性选择作用。
4．HCV准种的系统发育
对 EZ区序列的系统进化树分析显示，HCV 3b型重庆株与欧洲株的序
列差异较小，而比＊ 型各分离株间的序列差异较大；在持续性感染者中，
HCV 3b型毒株的同义碱基替换率明显低于 la及几型毒株。这提示HCV
不同亚型准种的演变程度有差异。
5．HOV结构蛋白在真核表达系统中的表达
将HCV C／EI／EZ基因长约Ikb片段亚克隆至哺乳动物表达载体，经
大肠杆菌 DHS a扩增，在 TNT／T7兔网织红细胞裂解物中表达，表达的蛋
白质分子量约57kDa。然而，随机逃?
Hepatitis C virus (HCV), a member of the Flavivirdate family is the major cause of chronic liver disease worldwide. HCV is a positive-sense single-strand RNA virus with a genome that encodes one large polyprotein in which putative structural proteins are located at the N-terminal end, and the putative nonstructural (NS) proteins are located at the C-terminal end. One of the important characteristics of HCV is that its genome exhibits significant genetic heterogeneity as a result of the accumulation of mutations during viral replication. The genetic sequences of HCV variants are very heterogeneous, varying by more than 30% across the entire genome among the six major genotypes, 20% among subtypes, and up to 10% within a subtype. Analogous to other RNA viruses, HCV circulates in an infected individual as a population of closed related, yet heterogeneous, sequences: the quasispecies. The quasispecies distribution of HCV might have important biological consequences. It has been proposed that this genetic heterogeneity allows HCV to escape immune pressure and to establish chronic infection. In addition, the existence of a heterogeneous population of HCV may influence the outcome of antiviral therapy; resistance to treatment might result from selection of minor viral populations during this therapy. Therefore, it is important to define accurately quasispecies populations of HCV.
Many analyses of viral quasispecies of HCV have been published. The majority of these studies have focused on the most variable part of the HCV
in
genome, hypervariable region 1 (HVR1) of glycoprotein E2. Mutation of this region of the genome is believed to be associated with viral persistence via immune escape mechanisms. However, it is well known that genetic heterogeneity of HCV extends throughout the entire genome. It is our hypothesis that significant mutation occurred in other regions of the viral envelope genes during the chronic infection. In addition, most studies assessing the diversity of HCV quasispecies are conducted by amplifying selected portions of the genome by PCR, isolating individual subgenomic fragments by a cloning produce, and then characterizing the nucleotide sequence of each clone. Evaluating the diversity of HCV quasispecies in clinical samples often requires the sequencing of a large number of clones, but because of the effort and expense, published studies obtain sequence information from a small number of colonies per subject.
In order to investigate genetic variation of HCV quasispecies and its relationship with the outcome of acute hepatitis C, HCV quasispecies were characterized in specimens collected every two to six months from a cohort of acutely HCV-infected individuals (the duration of specimen collection, 34 months after seroconversion). We evaluated 2 individuals who spontaneously cleared viremia and 3 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned El and the 5' half of E2, including HVR1. To assess the quasispecies complexity and to detect variants for sequencing and the expression of their functional proteins , Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined a single-stranded conformational polymorphism (SSCP) method and heteroduplex analysis (HD) analysis.
1. The mixed oligonucleotides primed amplification of the HCV structural region and Cloning of cDNA
The fragment spanning the 3' half of C region, El and 5' half of E2
IV
including HVR1 of Chinese viral strain with HCV type la> Ib or 3b, which was determined by RT-PCR and restriction fragment length polymorphism (RFLP) analysis of the 5' untranslated region (UTR), was amplified by using the mixed oligonucleotides primers so as to increase detective sensitivity toward variants in the plasma of an HCV infected patient. The 1 -kb amplicons were ligated into the vector pT-adv and used to transform E. coli TOP 10'F competent cells, in which cloning efficiency was > 90%.
2. Optimal screening of viral quasispeces in the structural region of HCV
For e
有关准种的研究近年来报道很多，但大多集中于HCV基因组变异最大的包膜2区(E2)N端的高度变异1区(HVR1)。研究表明，HVR1免疫逃逸株产生与病毒持续性感染有关。然而，研究证实，HCV异质性分布在整个基因组范围。持续性感染的HCV结构区可能还存在更多的有意义的变异区或位点。此外，准种研究的传统手段是分子克隆目标基因的PCR产物，并对每个克隆进行序列分析。要准确评估血标本中HCV准种的差异性，必须对大量克隆进行测序，但由于工作量大及开支高的原因，大多研究者仅对每例标本中的少量克隆进行测序。
本研究针对当前有关研究之不足，以急性HCV感染的两种转归即一过性及持续性感染者为对象，采用PCR扩增HCV结构区包括C区C端、
EI和EZ区N端长约 Ikb片段，并进行克隆，以单链构象多态性kingle strand
conformatlon polymorphism，SSCP）／异质性双体（heteroduplex，HD）分析为
准种筛选方法，分析了准种复杂性，并对所选的代表性准种进行核酸序列
分析及结构蛋白表达，以了解准种演变进化概貌及其与病情转归的关系。
1．HCV结构区基因的扩增及克隆
以5’NCR的限制性长度多态性分析为基因分型方法，采用混合碱基设
计的通用引物一次性扩增了 HCV la、fo及 3b型的 C肥用 区长约 Ikb片
断，以此提高准种检测的灵敏度。利用TA克隆载体PT－AdV对结构区基因
的P＊R产物进行克隆，使克隆效率＞90％。
2．HCV结构区准种的忧化筛选
选通过较长PCR扩增HCV结构区长约Ikb片断，继而分两组PCR
扩增 C／EI区 （47 hp）和 EZ／HVRI区 （57 hp），每仔病人随机选取一个克
隆的C／EI或EZ区扩增产物作为驱动子＊river卜 以SSCP用D方法对每份
标本的33个克隆的C用吧 区克隆型（准种）进行筛选。结果显示，每例
病人不同时相标本的EI 区克隆型存在廷续性；一过性感染者受感初期与
病毒清除前的EZ区优势克隆型虽不同，但未出现新的克隆型，而持续性感
染者急性期与漫性期血中的克隆型组成发生明显变化，并出现新的克隆型，
导致准种复杂性明显增加。
3．HCV准种的演变与急性感染的转归
对HCV一过性感染和持续性感染者不同时间标本的SSCP／HD优势克
隆型及劣势克隆型进行测序，通过序列差异性分析，观察HVRI及其他结
构区准种的演变状况；经第一时相对其他时相核酸序列的非同义碱基替换
和同义碱基替换率测定，进一步分析正性选择作用对准种演化的影响。结
果显示，持续性感染者中HVRI序列差异性明显增大，而一过性感染者则
无明显改变，两者的N及HVRI以外的EZ区序列差异性亦无明显差异，
表明一过性和持续性感染显示的准种演变形式不同主要由 HVR 变异所
致；持续性感染者HVRI的每月平均非同义碱基替换数呈增高趋势，两组
病人的m区和除外HVRI的EZ区＊02个氨基酸残基）平均非同义碱基
·IX·
、
替换数却保持低水平，相反，两组病人EI、HVRI及HVRI以外EZ区的
每月平均同义碱基替换数无显著差别，提示宿主兔疫压力主要对HVRI产
生阳性选择作用。
4．HCV准种的系统发育
对 EZ区序列的系统进化树分析显示，HCV 3b型重庆株与欧洲株的序
列差异较小，而比＊ 型各分离株间的序列差异较大；在持续性感染者中，
HCV 3b型毒株的同义碱基替换率明显低于 la及几型毒株。这提示HCV
不同亚型准种的演变程度有差异。
5．HOV结构蛋白在真核表达系统中的表达
将HCV C／EI／EZ基因长约Ikb片段亚克隆至哺乳动物表达载体，经
大肠杆菌 DHS a扩增，在 TNT／T7兔网织红细胞裂解物中表达，表达的蛋
白质分子量约57kDa。然而，随机逃?
Hepatitis C virus (HCV), a member of the Flavivirdate family is the major cause of chronic liver disease worldwide. HCV is a positive-sense single-strand RNA virus with a genome that encodes one large polyprotein in which putative structural proteins are located at the N-terminal end, and the putative nonstructural (NS) proteins are located at the C-terminal end. One of the important characteristics of HCV is that its genome exhibits significant genetic heterogeneity as a result of the accumulation of mutations during viral replication. The genetic sequences of HCV variants are very heterogeneous, varying by more than 30% across the entire genome among the six major genotypes, 20% among subtypes, and up to 10% within a subtype. Analogous to other RNA viruses, HCV circulates in an infected individual as a population of closed related, yet heterogeneous, sequences: the quasispecies. The quasispecies distribution of HCV might have important biological consequences. It has been proposed that this genetic heterogeneity allows HCV to escape immune pressure and to establish chronic infection. In addition, the existence of a heterogeneous population of HCV may influence the outcome of antiviral therapy; resistance to treatment might result from selection of minor viral populations during this therapy. Therefore, it is important to define accurately quasispecies populations of HCV.
Many analyses of viral quasispecies of HCV have been published. The majority of these studies have focused on the most variable part of the HCV
in
genome, hypervariable region 1 (HVR1) of glycoprotein E2. Mutation of this region of the genome is believed to be associated with viral persistence via immune escape mechanisms. However, it is well known that genetic heterogeneity of HCV extends throughout the entire genome. It is our hypothesis that significant mutation occurred in other regions of the viral envelope genes during the chronic infection. In addition, most studies assessing the diversity of HCV quasispecies are conducted by amplifying selected portions of the genome by PCR, isolating individual subgenomic fragments by a cloning produce, and then characterizing the nucleotide sequence of each clone. Evaluating the diversity of HCV quasispecies in clinical samples often requires the sequencing of a large number of clones, but because of the effort and expense, published studies obtain sequence information from a small number of colonies per subject.
In order to investigate genetic variation of HCV quasispecies and its relationship with the outcome of acute hepatitis C, HCV quasispecies were characterized in specimens collected every two to six months from a cohort of acutely HCV-infected individuals (the duration of specimen collection, 34 months after seroconversion). We evaluated 2 individuals who spontaneously cleared viremia and 3 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned El and the 5' half of E2, including HVR1. To assess the quasispecies complexity and to detect variants for sequencing and the expression of their functional proteins , Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined a single-stranded conformational polymorphism (SSCP) method and heteroduplex analysis (HD) analysis.
1. The mixed oligonucleotides primed amplification of the HCV structural region and Cloning of cDNA
The fragment spanning the 3' half of C region, El and 5' half of E2
IV
including HVR1 of Chinese viral strain with HCV type la> Ib or 3b, which was determined by RT-PCR and restriction fragment length polymorphism (RFLP) analysis of the 5' untranslated region (UTR), was amplified by using the mixed oligonucleotides primers so as to increase detective sensitivity toward variants in the plasma of an HCV infected patient. The 1 -kb amplicons were ligated into the vector pT-adv and used to transform E. coli TOP 10'F competent cells, in which cloning efficiency was > 90%.
2. Optimal screening of viral quasispeces in the structural region of HCV
For e
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43. Farci P, Alter HJ, Shimoda A, et al. Hepatitis C virus-associated fulminant hepatic failure. N Engl J Med, 1996, 335:631～634.
44. Brillanti S, Foli M, Gaiani S, et al. Persistent hepatitis C viremia without liver disease. Lancet, 1993, 341: 464～465.
45. Tanaka S, Takenaka K, Matsumata T, et al. Hepatitis C virus replication is associated with expression of transforming growth factor-alpha and insulin-like growth factor-Ⅱ in cirrhotic livers. Dig Dis Sci, 1996, 41: 208～215.
46. Gruber A, Lundberg LG, Bjorkholm M, et al Reactivation of chronic
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47. Liaw YF, Lee CS, Tsai SL, et al. T-cell-mediated autologous hepatocytotoxicity in patients with chronic hepatitis C virus infection. Hepatology, 1995, 22:1368～1373.
48. Minutello MA, Pileri P, Unutmaz D, et al. Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4+ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C. J Exp Med, 1993, 178: 17～25.
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68. Takahashi H, Merli S, PutneySD, et al. A single amino acid interchange yields reciprocal CTL specificities for HIV-1 gp160. Science, 1989, 246: 118～121.
69. Collins KL, Chen BK, Kalams SA, et al. HIV-1Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature, 1998, 391: 397～401.
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38. Farci P, Alter Hj, Govindarajan S, Wong DC, et al. Lack of protective immunity against reinfection with hepatitis C virus. Science, 1992, 258: 135～140.
39. Wyatt CA, Andrus L, Brotman B, et al. Immunity in chimpanzees chronically infected with hepatitis C virus: role of minor quasispecies in reinfection.
40. Zinkernagel RM, Haenseler E, Leist T, et al. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class Ⅰ-restricted virus-specific cytotoxic T cells as physiological correlate of the 51Cr-release assay? J Exp Med, 1986, 164: 1075～1092.
41. Morriyama T, Guilhot S, Klopchin K, et al. Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science, 1990, 248: 361～364.
42. Guidotti LG, Chisari FV. To kill or to cure: options in host defense against viral infection. Curr Opin Immunol, 1996, 8: 478～483.
43. Farci P, Alter HJ, Shimoda A, et al. Hepatitis C virus-associated fulminant hepatic failure. N Engl J Med, 1996, 335:631～634.
44. Brillanti S, Foli M, Gaiani S, et al. Persistent hepatitis C viremia without liver disease. Lancet, 1993, 341: 464～465.
45. Tanaka S, Takenaka K, Matsumata T, et al. Hepatitis C virus replication is associated with expression of transforming growth factor-alpha and insulin-like growth factor-Ⅱ in cirrhotic livers. Dig Dis Sci, 1996, 41: 208～215.
46. Gruber A, Lundberg LG, Bjorkholm M, et al Reactivation of chronic
hepatitis C after withdrawal of immunosuppressive therapy. J Intern Med, 1993, 234: 223～225.
47. Liaw YF, Lee CS, Tsai SL, et al. T-cell-mediated autologous hepatocytotoxicity in patients with chronic hepatitis C virus infection. Hepatology, 1995, 22:1368～1373.
48. Minutello MA, Pileri P, Unutmaz D, et al. Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4+ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C. J Exp Med, 1993, 178: 17～25.
49. Zinkernagel RM. Immunology taught by viruses. Science, 1996, 271: 173～178.
50. Lam NP, Neumann AU, Gretch DR, et al. Dose-dependent acute clearance of hepatitis C genotype 1 virus with interferon alfa. Hepatology, 1997, 26: 226～231.
51. Neumann Au, Lam NP, Dahari H, et al. Hepatitis C Viral dynamics in vive and the antiviral efficacy of interferon-alpha therapy. Science, 1998, 282: 103～107.
52. Franco A, Ferrari C, Sette A, et al. TCR antagonism and escape from the immune response. Curr Opin Immunol, 1995, 7:524～531.
53. Xu XN, Screaton GR, Gotch FM, et al. Evasion of cytotoxic T lymphocyte (CTL) responses by independent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells. J Exp Med, 1997, 186: 7～16.
54. Barry M, Mcfadden G. Virus encoded cytokines and cytokine receptors. Parasitology, 1997, 115: S89～S100,
55. Davis-Poynter NJ, Farrell HE, Masters of deception: a review of herpesvirus immune evasion strategies. Immunol Cell Biol, 1996, 74: 513～522.
56. Fleming SB, McCaughan CA, Andrews AE, et al. A homolog of interleukin-10 is encoded by the poxvirus or virus. J Virol, 1997, 71: 4857～4861.
57. Chen CM, You LR, Hwang LH, et al. Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-β receptor modulates the signal pathway of the lymphotoxin-β receptor. J Virol, 1997, 71:9417～9429.
58. Matsumoto M, Hsie T-Y, Zhu N, et al. Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-β receptor. J Virol, 1997, 71: 1301～1309.
59. Browning JL, Miatkowski K, Griffiths DA, et al. Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxin a and β. J Biol Chem, 1996, 271: 8616～8626.
60. Zhu N, Khoshnan A, Scheider R, et al. Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis. J Virol, 1998, 72: 3691～3697.
61. Ray RB, Meyer K, Shrivastava A, et al. Inhibition of tunor necrosis factor (TNF-alpha)-mediated apoptosis by hepatitis C virus core protein. J Biol Chem, 1998, 273: 2256～2259.
62. You LR, Chen CM, Lee YHW. Hepatitis C virus core protein enhances tumor necrosis factor alpha. J Virol, 1999, 73: 1672～1681.
63. Weiner AJ, Geysen HM, christopherson C, et al. Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infection. Proc Natl Acad Sci USA, 1992, 89: 3468～3472.
64. Polyak SJ, Faulkner G, Carithers RL, et al. Assessment of hepatitis C virus quasispecies heterogeneity by gel. Shift analysis: correlation with response to interferon therapy. J infect Dis, 1997, 175:1101～1107.
65. Oldstone MB. How viruses escapes from cytotoxic T lymphocytes: molecular parameters and players. Virology, 1997, 234:179～185.
66. McMichael AJ and Phillips RE. Escape of human immunodeficiency virus from immune control. Annu Rev Immunol, 1997, 15:271～296.
67. Shen L, Chen ZW, Letvin NL, et al. The repertoire of cytotoxic T lymphocytes in the recognition of mutat simian immunodeficiency virus variants. J Immunol, 1994, 153: 5849～5854.
68. Takahashi H, Merli S, PutneySD, et al. A single amino acid interchange yields reciprocal CTL specificities for HIV-1 gp160. Science, 1989, 246: 118～121.
69. Collins KL, Chen BK, Kalams SA, et al. HIV-1Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature, 1998, 391: 397～401.
70. Ho DD. Perspectives series: host/pathogen interactions. Dynamics of HIV-1 replication in vivo. J Clin Invest, 1997, 99: 2565～2567.
71. Weiner AJ, Erickson AL, Kansopon J, et al. Association of cytotoxic T lymphocyte (CTL) escape mutations with persistent hepatitis C virus (HCV) infection. Princess Takamatsu Symp, 1995, 25: 227～235.