In vitro maturation of human immunodeficiency virus type 1 Gag virus-like particle

人类免疫缺陷病毒1型Gag病毒样颗粒的体外成熟

基本信息

批准号：
11670309
负责人：
MORIKAWA Yuko
金额：
$ 2.3万
依托单位：
The Kitasato Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670309/
关键词：
HIV maturation processing in vitro particle infectivity

项目摘要

Human immunodeficiency virus (HIV) Gag proteins are assembled underneath the plasma membrane to form the budding virus particles. During or after budding, the particles undergo the process termed maturation in which Gag is cleaved by virion-containing HIV protease to yield the N-terminal matrix (MA), the central capsid (CA), the nucleocapsid (NC), the C-terminal p6 proteins. Concomitant with the processing, doughnut-like HIV particles (the immature form) are converted to particles containing condensed cores (the mature form). As the immature particles are non-infectious, the maturation process are essential for HIV infectivity. However, it is difficult to undestand nature of Gag processing in cell-based experiments as the process of virus particle budding and that of Gag processing are interlinked and neither process is not synchronized. To understand this process, we carried out the in vitro processing of immature HIV Gag virus-like particle (VLP) by exogenously added HIV protease. Fo … More llowing delipidization with Triton X-100, sequential processing of immature VLP was carried out in acidic or neutral buffers with different concentrations of salt and confirmed the optimum activity for HIV PR (mildly acidic pH and low salt concentration). Under all conditions tested, the MA/CA junction was cleaved faster than the CA/NC junction, an altered order of processing when compared with authentic processing. When the in vitro- processed VLP was analyzed on sucrose density gradients, most of MA, CA-p15 intermediate, and NC were detected as a highly multimeric form, equivalent to the unprocessed VLP.Reverse transcriptase, a processing product of the Pot region, was also found associated to the highly multimeric complex. In contrast, CA was found as a monomer dissociated from the multimeric CA-p15 following cleavage of the CA/NC junction. Electron microscopy revealed that the in vitro processing was accompanied by conversion of the doughnut-like particles to particles containing outer shells (which may correspond to a multimer of MA) and condensed cores (likely corresponding to a complex of NC and RNA). Chracteristic of most of the cores was the absence of core shells. These results imply that the processing order in VLP may be important for core shell formation but not outer shell or core formation. To examine whether in vitro processing of the immature particles leads to produce infectious particles, we initially explored a method by which the lipid bilayer of VLP was permeabilized without a loss of viral envelope protein.Immature pseudotype particles was prepared by co-transfection with HIV proviral DNA clone whose PR was inactive and env gene was replaced with the GFP gene and expression plasmid of vesicular stomatitis virus G protein. Following in vitro processing, the processed pseudotype particles were inoculated on 239T or HeLa cells. The level of GFP expression will clarify whether or not the in vitro-processed particles are capable to produce integration-competent replication complexes. Less

人类免疫缺陷病毒 (HIV) Gag 蛋白在质膜下组装，形成出芽病毒颗粒。出芽期间或出芽后，颗粒经历称为成熟的过程，其中 Gag 被含有病毒颗粒的 HIV 蛋白酶裂解，产生 N 末端。基质 (MA)、中央衣壳 (CA)、核衣壳 (NC)、C 末端 p6 蛋白。甜甜圈状的 HIV 颗粒（未成熟形式）转化为含有浓缩核心的颗粒（成熟形式），由于未成熟颗粒不具有感染性，因此成熟过程对于 HIV 的感染性至关重要。细胞实验中的Gag处理，因为病毒颗粒出芽的过程和Gag处理的过程是相互关联的，并且两个过程都不同步。为了理解这个过程，我们进行了未成熟病毒的体外处理。通过外源添加 HIV 蛋白酶，用 Triton X-100 减缓 HIV Gag 病毒样颗粒 (VLP) 的脱脂作用，在具有不同盐浓度的酸性或中性缓冲液中对未成熟的 VLP 进行连续处理，并确认了其最佳活性。 HIV PR（弱酸性pH和低盐浓度）在所有测试条件下，MA/CA连接处的裂解速度比CA/NC连接处快，与真实处理相比，处理顺序发生了变化。当对体外加工的VLP进行蔗糖密度梯度分析时，大多数MA、CA-p15中间体和NC被检测为高度多聚体形式，相当于未加工的VLP。逆转录酶是Pot区域的加工产物，还发现与高度多聚体复合物相关，相反，在 CA/NC 连接裂解后，发现 CA 作为单体从多聚体 CA-p15 中分离出来。体外加工伴随着甜甜圈状颗粒转化为含有外壳（可能对应于 MA 的多聚体）和凝聚核心（可能对应于 NC 和 RNA 的复合物）的颗粒。这些结果意味着 VLP 中的加工顺序可能对核壳的形成很重要，但对外壳或核心的形成并不重要。为了检查未成熟颗粒的体外加工是否会导致产生感染性颗粒，我们最初探索了一种方法。方法通过与PR失活的HIV原病毒DNA克隆共转染，用水疱性口炎病毒的GFP基因和表达质粒取代env基因，制备未成熟的假型颗粒。 G 蛋白。在体外处理后，将处理后的假型颗粒接种到 239T 或 HeLa 细胞上，GFP 表达水平将阐明是否是体外处理的。粒子能够产生具有整合能力的复制复合物。