EASL 2013: B肝病毒核糖核酸酶RNASEH，新的抗病毒药物开发目标

StephenW

Abstract 123
     
STRUCTURE OF THE HEPATITIS B VIRUS RNASE H, A TARGET FOR NEW ANTIVIRAL DRUG DEVELOPMENT, UNRAVELED BY ULTRA-DEEP PYROSEQUECING AND MOLECULAR MODELING
     
J. Hayer1, C. Rodriguez2*, G. Germanidis3, G. Deleage1, F. Zoulim4, J.-M. Pawlotsky2, C. Combet1
1IBCP, CNRS U5086, University Lyon 1, Lyon, 2Virology, INSERM Unit 955 Equip 18, Créteil, France, 3University of Thessaloniki, Thessaloniki, Greece, 4Viral Hepatitis Research Laboratory, U1052, INSERM; Lyon University Hospital, Lyon, France. *[email protected]

Last-generation nucleoside/nucleotide analogues are potent and have a high barrier to resistance. They help control viral replication in the short- to mid-term in a large proportion of treated patients. However, delayed responses have been observed in patients already exposed to other drugs of the same class, long-term resistance is possible, and cure of infection cannot be achieved with these therapies, emphasizing the need for other therapeutic approaches with different viral targets. Among them, the HBV RNase H represents an interesting target because its enzyme activity (cleavage of RNA/DNA heteroduplexes) is essential to the HBV life cycle. The goal of our study was to characterize the molecular structure of HBV RNase H. We generated a new predict 3D molecular model of HBV RNase H, derived from E. coli RNase H, using quasispecies sequences from patients infected with different HBV genotypes available in public database, and from a homogenous population of 73 treatment-naïve patients infected with HBV genotype D generated by means of ultra-deep pyrosequencing (454, Roche-Molecular-Systems). In the latter experiments, 958,000 sequences were generated, i.e. on average 12,900 sequences per patient, with an average sequence length of 302 base pairs. The new model revealed the following specificities of the HBV enzyme compared to other RNases H:
(i) among the 4 residues of the HBV RNase H catalytic site, one presents variability and additional one has been observed in one patient ; variability at this position is always silent in the overlapping HBx gene and appears to have no impact on the levels of viral replication;
(ii) The basic protrusion containing C-helix, which is required to guide the RNA/DNA heteroduplex into catalytic site, displays a highly conserved additional domain in regards to E. coli; this domain could be used to target RNAse H inhibitors that are specific for the HBV enzyme, without cross-species activity.
Conclusions: By means of ultra-deep pyrosequencing and molecular modeling, we have highlighted key features HBV RNAse H. The model shows substantial differences with other known RNases H, and paves the way for the development of new inhibitors of HBV cell cycle specifically targeting RNAse H activity.


Assigned speakers:
Mr. Christophe Rodriguez, INSERM U955Eq18, Paris-Est University , Créteil , France

Assigned in sessions:
27.04.2013, 15:30-17:30, Parallel Session, PS15, Parallel Session: HEPATITIS B & D EXPERIMENTAL, Hall 21

StephenW

摘要123

B型肝炎病毒核糖核酸酶H，新的抗病毒药物开发的目标，揭开超深PYROSEQUECING的和分子模拟的结构向后移动打印将该产品添加到您的行程

J. Hayer1 C. Rodriguez2 * G. Germanidis3，G. Deleage1，F. Zoulim4，J.-M. C. Combet1 Pawlotsky2
1IBCP，法国国家科学研究中心U5086，里昂大学里昂1，2Virology，INSERM的单位955装备18，克雷泰伊，法国，塞萨洛尼基3University，塞萨洛尼基，希腊，肝炎研究实验室，4Viral U1052，INSERM，里昂，法国里昂大学医院。 * christophe.rodriguez hmn.aphp.fr

最新代核苷/核苷酸类似物是强效的，并具有较高的障碍阻力。他们帮助控制病毒的复制，在短期到中期治疗的患者在一个大的比例。然而，延迟反应已经暴露在同一类的其他药物的患者中已经观察到，长期性是可能的，而且治愈的感染不能达到这些治疗，强调需要其他的治疗方法，不同的病毒目标。其中，的HBV RNA酶H代表一个有趣的目标，因为它的酶活性的（裂解RNA / DNA异源双链）的HBV生命周期是必不可少的。我们研究的目的是表征HBV RNA酶H。的分子结构，我们生成了新的预测HBV RNA酶H的三维分子模型，是来自于大肠杆菌核糖核酸酶H，使用准种序列从患者感染了不同的HBV基因型可在公共数据库，并从同质人口73治疗初治患者感染HBV D基因型所产生的超深的焦磷酸测序手段（454，罗氏分子系统）。 958000序列的产生在后面的实验中，每名患者平均12900序列，即，序列长度为302个碱基对的平均。新的模型揭示了以下的HBV特异性酶相比其他核糖核酸酶ħ：
（一）乙型肝炎病毒核糖核酸酶H催化部位之间的4个残基，呈现多变性和附加已观察到一个病人，在这个位置上的变化总是沉默在重叠的HBx基因，似乎没有影响水平病毒的复制;
（ii）本基本突起含有C-螺旋结构，这是需要引导到催化位点的RNA / DNA异源双链，显示一个高度保守的问候大肠杆菌额外的域，该域可用于目标RNA酶H抑制剂，特定的的HBV酶，不跨物种活动。
结论：通过超深的焦磷酸测序和分子模拟，我们强调主要功能，乙肝病毒核糖核酸酶H的模型显示与其他已知的RNA酶ħ的重大分歧，新的专门针对核糖核酸酶抑制HBV细胞周期的发展铺平了道路H活性。

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