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

人类免疫缺陷病毒(HIV)Gag蛋白在质膜下组装，形成萌芽中的病毒颗粒。在发芽过程中或发芽后，颗粒经历了一个称为成熟的过程，在这个过程中，GAG被含有病毒粒子的HIV蛋白酶切割，产生N端基质(MA)、中央衣壳(CA)、核衣壳(NC)和C端p6蛋白。随着加工的进行，甜甜圈状的艾滋病毒颗粒(未成熟的形式)被转化为含有凝聚核心的颗粒(成熟的形式)。由于未成熟的颗粒是非传染性的，成熟过程对艾滋病毒的传染性是必不可少的。然而，在基于细胞的实验中，很难理解GAG处理的本质，因为病毒颗粒发芽和GAG处理的过程是相互关联的，并且两个过程都不同步。为了了解这一过程，我们通过外源添加HIV蛋白酶对未成熟的HIV Gag病毒样颗粒(VLP)进行了体外加工。FO…用Triton X-100对未成熟的VLP进行了更缓慢的脱脂，并在不同盐浓度的酸性或中性缓冲液中进行了顺序处理，确定了对HIV PR的最佳活性(中等酸性和低盐浓度)。在所有测试的条件下，MA/CA结的切割速度快于CA/NC结，与真正的加工相比，加工顺序发生了变化。当对体外处理的VLP进行蔗糖密度梯度分析时，大多数MA、CA-p15中间体和NC被检测到为高度多聚体，相当于未处理的VLP。Pot区的加工产物反向转录酶也与高度多聚体复合体相关。相反，在CA/NC连接被切割后，CA被发现是从多聚体CA-p15解离的单体。电子显微镜显示，在体外处理过程中，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(可能对应于NC和RNA的络合物)的颗粒。大多数岩心的特征是没有核壳。这些结果表明，VLP中的加工顺序可能对核壳的形成很重要，但对外壳或核的形成并不重要。为了验证体外处理VLP的未成熟颗粒是否会产生感染性颗粒，我们初步探索了一种在不损失病毒包膜蛋白的情况下渗透VLP脂双层的方法，将PR失活的HIV前病毒DNA克隆与水泡性口炎病毒G蛋白的GFP基因和表达载体共转染成未成熟的伪型颗粒。体外处理后，将处理后的伪型颗粒接种到239T或HeLa细胞上。GFP的表达水平将阐明体外处理的颗粒是否能够产生整合能力强的复制复合体。较少

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