慢性1b型丙型肝炎病毒感染者包膜2基因正选择位点分析及功能意义

Positive selection analysis of hepatitis C virus genotype 1b envelope 2 gene and its significance during natural chronic infection

目的:纵向分析自然免疫压力下慢性1b型HCV感染者E2基因的系统进化模式及正选择位点,初步探讨E2基因正选择位点的分布与已知功能区之间的关系。方法:未抗病毒治疗的3例慢性1b型HCV感染者及其系列血清样本均来自中国人HCV感染患者系列标本库(CQueen cohort)。对每例感染者3个连续时间点的血清样本进行HCV5′端6000bp基因组的扩增、克隆,每个时间点随机挑选25～33个克隆对E2基因测序,用MEGA软件构建系统进化树,用固定效应似然法检测HCV E2基因的正选择位点。结果:3例HCV慢性感染者的9份血清样本均获清晰、无杂带的6030bp的目的片段。慢性HCV感染者体内病毒准种的复杂程度并非随着感染时间的延长而进行性增加,在某个时间点上准种变异株的序列可能会趋于一致。准种优势变异株在间隔半年时间以上已发生更替。对每位患者动态的准种变异株进行正选择位点的分析,共检测到5个氨基酸(aa)位点:aa384、aa399、aa410、aa475和aa522,分布于HCV E2基因HVR1区、HVR2区及HCV E2-CD81分子结合区。结论:基于动态变化的HCV准种优势株的相关研究,可考虑选择间隔半年以上时间点的血清样本作为研究对象。慢性HCV感染过程中,HCV E2基因位于重要功能区的aa位点受到了宿主强烈的免疫压力,aa384、aa399及aa410位点的变异可能导致HCV发生体液免疫逃逸;而aa475和aa522位点的变异则可能通过影响HVR2区的功能或HCV E2-CD81分子的亲和力或改变CD81分子的结构而导致HCV发生先天免疫逃逸。该研究为分析HCV C-NS3基因片段的正选择位点奠定了良好的基础。

AIM：To elucidate adaptive evolution patterns and perform a positive selection analysis of hepatitis C virus（HCV） genotype 1b envelope 2 gene（E2） during natural chronic infection by a longitudinal study. METHODS： HCV E2 quasispecies profiles were derived from partially sequenced domains of 6 000 bp recombinant clones. Phylogenetic trees for HCV E2 gene were constructed by MEGA software and the specific codons undergoing diversifying positive selection were identified by FEL method. RESULTS： HCV phylogenies, coupled with the number and distribution of selected sites were differed markedly between patients. HCV quasispecies complexity during chronic infection was not associated with the evolutionary time and the dominant viriant alternation of HCV quasispecies may occur after more than six months apart. Five sites under positive selection were identified within the ectodomain of the E2 protein. CONCLUSION： A series of serum specimens for studies based on dominant viriant of HCV dynamic quasispecies is recommended to be collected at every six months. Several individual sites of HCV E2 gene are under a strong host immune pressure, position aa384, aa399 and aa410 may be involved in escape from neutralizing antibodies, while position aa475, aa522 may correlate to modulate the virus-receptor interaction which result in evading immunity.