Interactions of Retroviral Proteins with Nucleic Acids

逆转录病毒蛋白之间似乎有几种不同的相互作用模式 和核酸，每一种都对病毒复制具有重要的功能性后果。第一、 Gag多聚蛋白（Gag的结构蛋白）的精确特异性识别。 病毒颗粒）在病毒组装期间选择用于包装的病毒RNA。的相互作用 具有RNA的逆转录病毒Gag蛋白非常复杂。我们认为， 病毒RNA是将Gag蛋白紧密结合在一起，正如我们在 逆转录病毒组装和成熟（ZIA BC 010511）。同时，成功的复制 需要病毒基因组RNA（vRNA），其含有包装信号（称为“Psi”）， 选择优先于成千上万的细胞RNA种类进行腺苷酸化， 也是用于包装的潜在基底。最后，HIV-1 Gag及其裂解产物 核衣壳（NC）具有核酸伴侣活性，使它们能够催化 核酸重排为具有最低自由能的构象。似乎 Gag中的这种活性负责将tRNA分子退火为vRNA， NC的伴侣蛋白活性在逆转录过程中起几个关键作用。我们 研究致力于在分子水平上阐明这些不同的相互作用。到 为了更好地了解HIV-1 RNA包装和组装之间的关系，我们正在研究 以下问题：HIV-1 Gag如何区分不同的RNA，以及如何 我们解释了在粒子组装过程中含有Psi的RNA的选择性包装？_我们 已经发现，当Gag在缺乏vRNA的情况下在体内表达时，它几乎可以包装 任何细胞mRNA。类似地，向HIV-1 Gag添加几乎任何核酸都会导致 病毒样颗粒（VLP）的体外组装。因此，vRNA与mRNA竞争， 封装; Psi封装信号使其在竞争中具有优势。我们正在研究 利用荧光技术研究重组Gag与含Psi的RNA和对照RNA的结合 相关光谱、微尺度热泳、SwitchSense仪器和质谱 测光法我们发现，Gag合作结合RNA。加格对超能力的亲和力 略高于对照RNA。使用几个Gag突变体，我们发现， 与对照的结合主要归因于基质结构域。值得注意的是，与Psi的结合 具有不同的特性，因为它比与对照RNA结合更耐盐， 表示较高的非静电分量。我们正在测试绑定到Psi的想法 触发了Gag经历的构象转变，使其更多地处于组装就绪状态 比其他RNA更有效。我们已经发现，结合到Psi支持粒子组装更多 与其他RNA结合的效率。这可以解释vRNA的选择性包装。的 数据还鉴定了Psi中的特定核苷酸， 粒子组装__与本项目相关的专利：美国专利#5，674，720：“设计和 基因组RNA缺陷的非感染性人逆转录病毒突变体的构建”; 1997年10月7日; Robert J. Gorelick，Larry O.放大图片作者：亚瑟，艾伦.亨德森，以及 斯蒂芬·奥罗斯兰。该专利描述了HIV-1的突变体，其结构正常，但 无感染性;这些突变体可能被认为是疫苗成分。美国 专利#7，572，828：“鉴定抑制病毒组装和结合的抗HIV化合物” of Nucleocapsid Protein to Nucleic Acid”; issued August 11，2009; Robert Shoemaker，Michael Currens，Alan Rein，Ya-Xiong Feng，Robert Fisher，Andrew Stephen，Shizuko Sei，布鲁斯克里斯， 路易斯亨德森和凯伦沃西。该专利描述了一类具有抗HIV-1 活动，正在调查用于抗逆转录病毒疗法。

There appear to be several different modes of interaction between retroviral proteins and nucleic acids, each with important functional consequences for viral replication. First, an exquisitely specific recognition by the Gag polyprotein (the structural protein of the virus particle) selects the viral RNA for packaging during virus assembly. The interactions of retroviral Gag proteins with RNAs are remarkably complex. We believe that one role played by the viral RNA is to bring Gag proteins close together, as discussed in our project on Retrovirus Assembly and Maturation (ZIA BC 010511). At the same time, successful replication requires that viral genomic RNA (vRNA), which contains a packaging signal (termed "Psi"), be selected for encapsidation in preference to the thousands of cellular RNA species that are also potential substrates for packaging. Finally, both HIV-1 Gag and its cleavage product nucleocapsid (NC) possess nucleic acid chaperone activity, enabling them to catalyze rearrangements of nucleic acids to the conformation with the lowest free energy. It appears that this activity in Gag is responsible for annealing a tRNA molecule to vRNA, and the chaperone activity of NC plays several critical roles during reverse transcription. Our research is devoted to elucidating these diverse interactions at the molecular level. To better understand the relationship between HIV-1 RNA packaging and assembly, we are addressing the following questions: How does HIV-1 Gag discriminate between different RNAs, and how can we explain the selective packaging of Psi-containing RNA during particle assembly? _____ We have found that when Gag is expressed in vivo in the absence of vRNA, it can package almost any cellular mRNA. Similarly, addition of almost any nucleic acid to HIV-1 Gag will lead to virus-like particle (VLP) assembly in vitro. Thus, vRNA is in competition with mRNAs for packaging; the Psi packaging signal gives it an advantage in this competition. We are studying the binding of recombinant Gag to Psi-containing and control RNAs by using fluorescence correlation spectroscopy, microscale thermophoresis, SwitchSense instrumentation, and mass photometry. We find that Gag binds RNAs cooperatively. The affinity of Gag for Psi is only modestly higher than that for control RNA. Using several Gag mutants, we have found that binding to the control is largely attributable to the matrix domain. Notably, binding to Psi has a different character, as it is far more salt resistant than binding to control RNA, indicating a higher nonelectrostatic component. We are testing the idea that binding to Psi triggers the conformational shift that Gag undergoes to an assembly-ready state more efficiently than other RNAs. We have found that binding to Psi supports particle assembly more efficiently than binding to other RNAs. This would explain selective packaging of vRNA. The data also identify specific nucleotides within Psi that are required for this efficient particle assembly. _____Patents linked to this project: U.S. Patent #5,674,720: "Design and Construction of Noninfectious Human Retroviral Mutants Deficient in Genomic RNA"; issued October 7, 1997; Robert J. Gorelick, Larry O. Arthur, Alan Rein, Louis E. Henderson, and Stephen Oroszlan. This patent describes mutants of HIV-1 that are structurally normal but noninfectious; these mutants could potentially be considered as vaccine constituents. U.S. Patent #7,572,828: "Identification of Anti-HIV Compounds Inhibiting Virus Assembly and Binding of Nucleocapsid Protein to Nucleic Acid"; issued August 11, 2009; Robert Shoemaker, Michael Currens, Alan Rein, Ya-Xiong Feng, Robert Fisher, Andrew Stephen, Shizuko Sei, Bruce Crise, Louis Henderson, and Karen Worthy. This patent describes a class of compounds with anti-HIV-1 activity, which are under investigation for use in antiretroviral therapy.