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: 10326908
• 负责人: Sebla B. Kutluay
• 金额: $ 23.63万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326908
• 关键词: 5' Untranslated Regions; Adenosine; Affect; Affinity; Antiviral Agents; 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; Structure; System; Testing; Therapeutic; Vaccines; 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; 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.

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 由病毒和细胞RNA结合蛋白（RBP）调节，这些蛋白识别不同的序列或结构， 病毒RNA的特征。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 Gag将两个拷贝的未剪接的正链病毒基因组包装在颗粒中， 选自过量的细胞和剪接的病毒mRNA库。病毒基因组是如何被选择的 包装以及为什么只有两个拷贝包装在一个病毒颗粒中仍然知之甚少。 从简单的逆转录病毒，如鼠白血病病毒（MLV）的发现延伸，长期以来， 认为HIV-1选择性基因组包装类似地由顺式作用包装信号Psi（Psi）调节， 位于基因组的5'非翻译区。然而，与MLV不同的是， 适度地影响包装，并且在DNA序列之外的区域可能有助于基因组的扩增。 因此，对HIV-1选择性基因组包装的精确规则仍然知之甚少。HIV-1 一个基因组有一个异常偏向的核苷酸组成，富含腺苷（~36%），而缺乏胞嘧啶 （~18%）。我们先前已经发现，Gag与HIV-1基因组的结合是高度动态的， 一些变化与其膜结合、多聚化和蛋白水解成熟一致。特别是， 我们发现，虽然胞质Gag与富含鸟苷的序列结合，但其结合偏好向 在质膜上具有富含腺苷的核苷酸组合物的序列伴随其 多聚化 在这个应用中，我们提出测试新的想法，即HIV-1基因组的总核苷酸含量 有助于其选择性包装。我们将进行一系列的遗传学研究， (NC)Gag的结构域被异源RNA结合结构域取代，并通过用 其中富含A与缺乏A核苷酸含量的最小基因组被包装成病毒体（Aim 1）。通过 用异源RNA结合结构域替换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是由Gag的特异性、亲和力和亲合力驱动的， 针对病毒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.理解这一过程不仅从基本的分子生物学观点来看是重要的， 也影响了基因治疗和基于CRISPR的工程工具，这些工具依赖于逆转录病毒系统进行有效的 递送到宿主细胞中。