Molecular mechanism of selective HIV-1 genome packaging

摘要 事实上，HIV-1复制的每一步以及许多细胞抗病毒防御机制都是 受识别不同序列或结构的病毒和细胞RNA结合蛋白(RBPs)调控 病毒RNA上的特征。其中一种相互作用发生在HIV-1主要结构蛋白GAG和 病毒基因组RNA。GAG将未剪接的正链病毒基因组的两个副本包装在颗粒中，这 是从过量的细胞和剪接病毒mRNA池中挑选出来的。病毒基因组是如何被选择的 包装和为什么只有两个拷贝被包装在一个病毒颗粒中仍然知之甚少。 通过推广简单逆转录病毒的发现，如小鼠白血病病毒(MLV)，长期以来 认为HIV-1选择性基因组包装类似地受到顺式作用包装信号Psi(Ψ)的调控， 位于基因组的5‘非翻译区。然而，与MLV不同的是，仅破坏Ψ中的区域 对包装有一定影响，Ψ序列以外的区域可能有助于基因组封装。 因此，管理HIV-1选择性基因组包装的确切规则仍然知之甚少。HIV-1 基因组具有异常偏向的核苷酸组成，富含腺苷(~36%)，贫胞苷 (~18%)。我们以前已经发现，Gag与HIV-1基因组的结合是高度动态的，并经历了 一些变化与其膜结合、多聚化和蛋白水解性成熟相一致。特别是， 我们发现，当胞质中的Gag与富含鸟苷的序列结合时，其结合偏好向 伴随ITS的质膜富含腺苷核苷酸组成的序列 多聚合化。 在这个应用中，我们建议测试新的想法，即HIV-1基因组的总核苷酸含量 有助于它的选择性包装。我们将进行一系列遗传方法，在这些方法中，核衣壳 GAG的(NC)结构域被异源RNA结合域取代，并通过测定与 A丰富与A贫乏核苷酸含量的最小基因组被包装成病毒粒子(目标1)。穿过 将NC替换为异源RNA结合域并增加每个GAG分子的NC拷贝数， 我们建议确定二聚体基因组包装是否由Gag的特异性、亲和力和亲和力驱动 走向病毒RNA(目标2)。总体而言，该项目将为选择性基因组的机制提供新的见解。 包装。了解这一过程不仅从基本的分子生物学角度具有重要意义，而且将 也影响基因治疗和基于CRISPR的工程工具，这些工具依赖于逆转录病毒系统来实现高效 运送到宿主细胞中。

Abstract Virtually every step of HIV-1 replication as well as numerous cellular antiviral defense mechanisms are regulated by viral and cellular RNA-binding proteins (RBPs) that recognize distinct sequence or structural features on viral RNAs. One such interaction takes place between the HIV-1 major structural protein, Gag, and the viral genomic RNA. Gag packages two copies of an unspliced positive strand viral genome in particles, which are selected from a pool of cellular and spliced viral mRNAs in excess. How viral genomes are selected for packaging and why only two copies are packaged in a single virus particle remain poorly understood. By extension of findings from simple retroviruses, such as murine leukemia virus (MLV), it has long been thought that HIV-1 selective genome packaging is similarly regulated by a cis-acting packaging signal, Psi (Ψ), located within the 5’ untranslated region of the genome. However, unlike MLV, disruption of regions in Ψ only modestly impacts packaging and regions outside of the Ψ sequence might contribute to genome encapsidation. Thus the precise rules that govern HIV-1 selective genome packaging still remain poorly understood. HIV-1 genome has an unusually biased nucleotide composition, rich in adenosines (~36%) and poor in cytosines (~18%). We have previously discovered that Gag binding to the HIV-1 genome is highly dynamic and undergoes several changes coincident with its membrane binding, multimerization, and proteolytic maturation. In particular, we found that while cytosolic Gag bound to guanosine-rich sequences, its binding preference shifted towards sequences with adenosine-rich nucleotide composition at the plasma membrane concomitant with its multimerization. In this application, we propose to test the novel idea that the overall nucleotide content of the HIV-1 genome contributes to its selective packaging. We will conduct a series of genetic approaches in which the nucleocapsid (NC) domain of Gag is replaced by heterologous RNA-binding domains and by determining the efficiency with which minimal genomes with A-rich vs. A-poor nucleotide content are packaged into virions (Aim 1). Through replacement of NC by heterologous RNA-binding domains and increasing NC copy number per Gag molecule, we propose to determine whether dimeric genome packaging is driven by specificity, affinity and avidity of Gag towards viral RNAs (Aim 2). Overall, this project will provide novel insight into mechanisms of selective genome packaging. Understanding this process is not only significant from a basic molecular biology standpoint but will also impact gene therapy and CRISPR-based engineering tools which depend on retroviral systems for efficient delivery into host cells.