Molecular mechanism of selective HIV-1 genome packaging

HIV-1基因组选择性包装的分子机制

• 批准号: 10409845
• 负责人: Sebla B. Kutluay
• 金额: $ 19.69万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-24 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10409845
• 关键词: 5' Untranslated Regions; Adenosine; Affect; Affinity; Antiviral Therapy; Avidity; Binding; Biochemical; Biological Assay; Cell membrane; Cells; Chimera organism; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Complement; Complex; Cytosine; Cytosol; Defect; Defense Mechanisms; Dependence; Development; Engineering; Ensure; Equilibrium; Genetic Variation; Genome; Guanosine; HIV; HIV Genome; HIV-1; HIV-1 integrase; Membrane; Messenger RNA; Microscopy; Modeling; Molecular; Molecular Biology; Murine leukemia virus; Northern Blotting; Nucleocapsid; Nucleotides; Play; Process; Property; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Retroviridae; Role; Series; Signal Transduction; Site; Specificity; Structural Protein; System; Testing; Therapeutic; Viral; Viral Genome; Virion; Virus; Zinc Fingers; base; dimer; gag Gene Products; gene therapy; genetic approach; genetic regulatory protein; genomic RNA; insight; mutant; novel; particle; preference; recruit; sensor; tool; vaccine strategy; viral RNA; viral genomics; virtual

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.

抽象的 实际上，HIV-1复制的每个步骤以及许多蜂窝病毒防御机制是 由病毒和细胞RNA结合蛋白（RBP）调节，这些蛋白（RBP）识别不同的序列或结构 病毒RNA的功能。一种这样的相互作用发生在HIV-1主要结构蛋白，GAG和 病毒基因组RNA。插入颗粒中未填充的阳性链病毒基因组的两个副本 从过量的细胞和剪接病毒mRNA中选择。如何选择病毒基因组 包装以及为什么在单个病毒颗粒中只包装两份副本的原因仍然很少。 通过扩展来自简单逆转录病毒的发现，例如鼠白血病病毒（MLV），长期以来一直是 认为HIV-1选择性基因组包装受到顺式作用包装信号PSI（ψ）， 位于基因组的5'未翻译区域内。但是，与MLV不同，仅在ψ中破坏区域 适度影响包装和ψ序列之外的区域可能有助于基因组封装。 控制HIV-1选择性基因组包装的确切规则仍然知之甚少。 HIV-1 基因组具有异常有偏的核苷酸组成，富含腺苷（〜36％），细胞学较差 （〜18％）。我们以前已经发现，与HIV-1基因组的插科打结合是高度动态的，并且经历了 几种变化与其膜结合，多其中临化和蛋白水解成熟的重合。尤其， 我们发现，虽然胞质堵塞结合到鸟嘌呤富序列，但其结合偏好转向 质膜伴有富含腺苷的核苷酸组成的序列与其序列 多聚化。 在此应用中，我们建议测试HIV-1基因组的整体核苷酸含量的新思想 有助于其选择性包装。我们将进行一系列遗传方法 （NC）GAG的结构域被异源RNA结合结构域取代，并通过确定效率 哪些具有富含A的基因组与贫穷的核苷酸含量被包装到病毒中（AIM 1）。通过 通过异源RNA结合域替换NC，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插科打的特异性，亲和力和亲和力驱动 病毒RNA（AIM 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。从基本的分子生物学的角度来看，了解这一过程不仅很重要，而且将 还影响基因疗法和基于CRISPR的工程工具，这些工具依赖于逆转录病毒系统以提高效率 输送到宿主细胞中。