乙型肝炎病毒聚合酶新功能区的发现和意义研究

发布时间：2018-05-06 15:03

本文选题：乙型肝炎病毒 + 人免疫缺陷病毒　；参考：《复旦大学》2007年博士论文

【摘要】： 乙型肝炎病毒聚合酶新功能区的发现和意义研究乙型肝炎病毒(Hepatitis B virus，HBV)感染在全球超过3亿人。HBV属于嗜肝DNA病毒科，该科具有部分双链环状的DNA基因组。HBV通过特有的逆转录反应进行复制。HBV编码的聚合酶／逆转录酶(Reverse transcriptase，RT)的结构和功能在其复制过程中起重要作用。但是，至今具有酶活性的HBV RT仍不能足量表达用于体外生化研究和晶体学分析。此外，也没有合适的体外感染系统用于分析复杂的HBV复制周期。通过对分离自乙肝病人的两株B基因型HBV的全基因组克隆和基于细胞转染的复制效率测定，我们曾报道HBV RT第306位脯氨酸(rtP306)置换为丝氨酸或其他氨基酸时，大部分变异体的复制效率下降。为阐明这些变异体复制效率下降的机理，我们首先构建一C基因型毒株的rtP306变异体并转染Huh-7细胞，然后比较变异体与原毒株的复制能力、转录水平和pgRNA包装效率。通过基于细胞转染的复制效率测定发现rtP306变异体复制效率下降，而且RT变异体与RT缺失复制子rtW58~*的反式补充实验也证实这一现象。为深入了解复制效率下降的机理，首先分析变异体和原毒株的转录水平，结果显示各rtP306变异体具有相似的转录水平。而且Western印迹显示rtP306氨基酸置换并不影响RT稳定性。我们通过特异性的pgRNA包装实验和内源性聚合酶反应分析HBV RT的两大主要功能：参与pgRNA包装和催化核苷转移合成子代病毒DNA。pgRNA包装效率用细胞内衣壳颗粒量校正后的衣壳颗粒内pgRNA量表示，细胞内衣壳颗粒用抗衣壳蛋白抗体进行Western印迹测定。包装实验结果显示大部分rtP306变异体的pgRNA包装效率下降。这一结果也被内源性聚合酶反应所确认。同时，通过内源性聚合酶反应发现一些rtP306氨基酸置换导致RT核苷转移酶活性下降。基于上述发现，我们推测除了rtP306外，rtP306附近的氨基酸(rt304-311)对HBV复制同样重要。通过将rt304-311位氨基酸置换为丙氨酸或苯丙氨酸构建变异体并测定其复制效率，我们发现rt304-311置换确实导致HBV复制效率明显下降。相关机理不是转录水平或RT稳定性改变而是pgRNA包装效率下降。作为对照，远离rtP306的氨基酸(rt336和rt346)置换不影响病毒复制效率。据我们所知，这是首次报道rt304-311氨基酸保守在pgRNA包装中起重要作用。此外，与原毒株相比，rtY305A变异体对抗病毒药阿德福韦的敏感性下降。阿德福韦据报道作用于HBV RT“手掌”域的酶活性中心，而rtY305置换位于HBV RT“拇指”域，因此药敏实验提示rt304-311氨基酸置换可能改变HBV RT构型从而导致与抗病毒药的亲和力下降。此外，通过生物信息学方法分析rt304-311对HBV RT和HBV复制的作用，结果显示：1)预测rt304-311位于HBV RT“拇指”域一螺旋-转角-螺旋结构的转角上，这一螺旋-转角-螺旋结构称为螺旋钳基序；2)转角氨基酸在已报导的基因型A-H毒株中非常保守；3)部分HBV RT转角氨基酸在逆转录病毒RT和丙型肝炎病毒RNA依赖的RNA聚合酶的螺旋钳基序中保守。螺旋钳基序是真核生物、细菌和病毒的核酸聚合酶拇指域中一共同的结构元件，在聚合反应时可结合核酸模板和引物。因此，我们通过生物学和结构生物信息学方法确认了HBV RT螺旋钳基序转角(rt304-311)，该转角作为一新功能区可能通过控制HBV RT与核酸的亲和力来调节HBV pgRNA包装效率。人免疫缺陷病毒(Human immunodeficiency virus，HIV)是逆转录病毒科慢病毒属的研究模型。与HBV相似，HIV也通过特有的逆转录反应进行复制。为研究HIV RT螺旋钳基序转角是否在HIV复制中起重要作用，以一HIV前病毒质粒基础上将HIV RT转角置换为丙氨酸。通过单周期感染实验检测各变异体的复制能力。初步结果显示HIV-1转角变异体(rtY271A和rtl274A)不能在Jurkat细胞内复制。而且，HIV-1 RT转角变异体(rtY271A和rtl274A)与DNA模板／引物结合能力下降。总之，HBV RT和HIV RT螺旋钳基序转角在病毒复制过程中起重要作用并且可以作为抗病毒药物设计的新靶点。目前，已注册的抗HBV药物如拉米呋啶，阿德福韦酯和恩替卡韦都是作用在HBV RT催化中心的核苷(酸)类似物。长期使用这些药物将导致耐药株的出现。因此迫切需要发展新的抗HBV药物。在筛选针对HBV RT和HIV RT螺旋钳基序转角的化合物时，我们发现一种非核苷类似物KM-1，2-萘磺酸，4-羟基-7-[[[[5-羟基—6-[(4-肉桂酸苯基)偶氮]-7-磺酸-2-萘基]氨基]羰基]氨基]-3-[(4-肉桂酸苯基)]偶氮(2-naphthalenesulfonic acid，(4-hydroxy-7-[[[[5-hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)]azo))曾被报道通过与已知非核苷类似物不同的机制有效地抑制HIV RT。而且，据推测，，KM-1针对HIV RT的“拇指”域。因此，我们采用多种方法评价KM-1的抗HBV活性。通过内源性聚合酶反应发现KM-1抑制野生型HBV RT及其对三磷酸拉米呋啶耐药的变异体rtL180M／M2041，50％抑制浓度分别为2.6μM和0.5μM，因此KM-1通过与三磷酸拉米呋啶不同的机制抑制HBV RT。通过基于细胞转染的复制效率测定发现KM-1抑制野生型HBV及其变异体rtL180M／M2041在细胞内复制，50％抑制浓度分别为31.2μM和9.5μM。此外，KM-1有一定细胞毒性，50％细胞毒浓度为105μM。因此，设计能与RT拇指区甚至拇指区螺旋钳基序转角结合的新化合物有望发展成新的抗HBV或抗HIV药物。
[Abstract]:Discovery and significance of new functional region of hepatitis B virus polymerase
Hepatitis B virus (Hepatitis B virus, HBV) infection in more than 300 million people all over the world.HBV belongs to the liver DNA family, the family has a partial double chain ring DNA genome.HBV that replicates the structure and function of the.HBV encoded polymerase / reverse transcriptase (Reverse transcriptase, RT) in its replication process. However, the HBV RT with enzyme activity still cannot be expressed enough for biochemical and crystallographic analysis in vitro. In addition, there is no suitable external infection system for analyzing complex HBV replication cycles. Complete genome cloning of two B genotype HBV isolated from hepatitis B patients and the replication efficiency based on cell transfection Rate determination, we reported that when HBV RT 306th bits of proline (rtP306) was replaced by serine or other amino acids, the replication efficiency of most of the variants decreased. In order to clarify the mechanism of the decrease in the replication efficiency of these variants, we first constructed a rtP306 variant of a C genotype and transfected Huh-7 cells, and then compared the variant and the original strain. Replication ability, transcriptional level and pgRNA packaging efficiency. The replication efficiency of rtP306 variants was detected by cell transfection based replication efficiency, and a trans supplemental experiment with RT variant and RT deletion replicon rtW58~* also confirmed this phenomenon.
In order to understand the mechanism of replication efficiency decline, the transcriptional level of the variant and the original strain was analyzed. The results showed that the rtP306 variants had similar transcriptional levels. And the Western blot showed that the rtP306 amino acid replacement did not affect the stability of RT. We analyzed HBV R by specific pgRNA packaging experiments and endogenous polymerase chain reaction. The two main functions of T: participation in pgRNA packaging and catalytic nucleoside transfer synthesis of subgeneration virus DNA.pgRNA packaging efficiency using the pgRNA quantity in the capsid particles adjusted by the cell lingerie shell particle size, and the cell lingerie shell particles using the anti capsid protein antibody for Western imprinting. The packaging test results show the pgRNA of most of the rtP306 variants. This result was also confirmed by endogenous polymerase reaction. At the same time, some rtP306 amino acid replacement resulted in the decrease of RT nucleoside transferase activity by endogenous polymerase reaction.
Based on the above findings, we speculate that in addition to rtP306, the amino acid (rt304-311) near rtP306 is equally important for HBV replication. By replacing rt304-311 - based amino acids to alanine or phenylalanine to construct variants and determining their replication efficiency, we found that rt304-311 replacement did lead to a significant decline in the efficiency of HBV replication. The related mechanism is not a turn. Recording level or RT stability change is the decrease in pgRNA packaging efficiency. As a control, the removal of rtP306 amino acids (rt336 and rt346) does not affect viral replication efficiency. To our knowledge, this is the first report that the rt304-311 amino acid conservatism plays an important role in pgRNA packaging. In addition, the rtY305A variant is against the virus drug A De compared to the original strain. The sensitivity of fovir declined. Adefovir was reported to be used as an enzyme activity center in the HBV RT "palm" domain, while rtY305 replacement was located in the HBV RT "thumb" domain, so the drug sensitivity experiment suggested that the rt304-311 amino acid replacement might change the HBV RT configuration and thus lead to a decrease in affinity with the antiviral drugs.
In addition, the effect of rt304-311 on HBV RT and HBV replication is analyzed by bioinformatics. The results show that: 1) the prediction of rt304-311 is located at the corner of a spiral angle spiral structure in the HBV RT "thumb" domain, and this spiral angle spiral structure is called the spiral forceps, and 2) the angular amino acids are very guaranteed in the reported genotype A-H strain. Guard; 3) partial HBV RT corner amino acids are conserved in the spiral forceps of retrovirus RT and RNA polymerase of hepatitis C virus RNA dependent. The spiral forceps are a common structural element in the nucleic acid polymerase of the eukaryotic, bacterial and viral nucleic acid polymerase in the thumb domain, which can be combined with nucleic acid templates and primers during polymerization. Biological and structural bioinformatics methods confirm the HBV RT spiral forceps base sequence angle (rt304-311), which may regulate the HBV pgRNA packaging efficiency by controlling the affinity of HBV RT with the nucleic acid as a new functional area.
Human immunodeficiency virus (HIV) is a study model of the genus lentivirus in the retroviral family. Similar to HBV, HIV also replicates through specific reverse transcriptase. To study whether the HIV RT spiral forceps base sequence angle plays an important role in HIV replication, and the HIV RT transfer angle is replaced by a HIV virus plasmid. The replication ability of various variants was detected by a single cycle infection test. Preliminary results showed that HIV-1 angle variants (rtY271A and rtl274A) could not be replicated in Jurkat cells. Moreover, the binding ability of HIV-1 RT angle variants (rtY271A and rtl274A) to DNA template / primers decreased. In a word, HBV RT and HIV spiral forceps were in the virus. Replication plays an important role and can be used as a new target for antiviral drug design.
Currently, registered anti HBV drugs such as lamamoxurin, adefovir and entecavir are all nucleoside (acid) analogues that act at the HBV RT catalytic center. Long term use of these drugs will lead to the emergence of resistant strains. Therefore, the development of new anti HBV drugs is urgently needed. When screening compounds for HBV RT and HIV RT spiral forceps, I We found a non nucleoside analogue, KM-1,2- naphthalene sulfonic acid, 4- hydroxyl -7-[[[[5- hydroxyl 6-[(4- cinnamic acid phenyl) azo]-7- sulfonic acid -2- naphthyl] carbonyl] amino]-3-[(4- cinnamate phenyl)] AZO (2-Naphthalenesulfonic acid, 4-hydroxy-7-[[[[5-hydroxy-6-[) ]-3-[(4-cinnamylphenyl)]azo)) has been reported to effectively inhibit HIV RT. through a mechanism different from the known non nucleoside analogues and it is presumed that KM-1 is directed to the "thumb" domain of HIV RT. Therefore, we have used a variety of methods to evaluate the anti HBV activity of KM-1. The KM-1 inhibitory wild type HBV, and its three phosphoric acid, were found by the endogenous polymerized enzyme reaction. The inhibitory concentrations of lamifuridine resistant variants rtL180M / M2041,50% were 2.6 mu M and 0.5 M respectively. Therefore, KM-1 inhibited HBV RT. by inhibiting the replication efficiency based on cell transfection by the different mechanisms of lamifuridine phosphate three, and found that KM-1 inhibited wild type HBV and its variant rtL180M / M2041 in cell replication and 50% inhibition concentration. In addition to 31.2 M and 9.5 mu M., KM-1 has a certain cytotoxicity and the concentration of 50% cells is 105 M., so a new compound designed to combine with the RT thumb region and even the spiral forceps of the thumb area is expected to develop into a new anti HBV or anti HIV drug.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373

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