Interactions of Retroviral Proteins with Nucleic Acids

逆转录病毒蛋白和核酸之间似乎有几种不同的相互作用模式，每种模式对病毒复制都有重要的功能影响。首先，Gag多蛋白（病毒颗粒的结构蛋白）在病毒组装过程中进行精细的特异性识别，选择病毒RNA进行包装。逆转录病毒Gag蛋白与rna的相互作用非常复杂。我们认为病毒RNA的一个作用是将Gag蛋白聚集在一起，正如我们在逆转录病毒组装和成熟项目（ZIA BC 010511）中所讨论的那样。与此同时，成功的复制需要病毒基因组RNA (vRNA)，它包含一个包装信号（称为“Psi”），被选择用于封装，而不是成千上万的细胞RNA物种，它们也是潜在的包装底物。最后，HIV-1 Gag及其切割产物核衣壳（NC）都具有核酸伴侣活性，使它们能够催化核酸重排到自由能最低的构象。似乎Gag中的这种活性负责将tRNA分子退火为vRNA，而NC的伴侣活性在逆转录过程中起着几个关键作用。我们的研究致力于在分子水平上阐明这些不同的相互作用。为了更好地理解HIV-1 RNA包装和组装之间的关系，我们正在解决以下问题：HIV-1 Gag如何区分不同的RNA，以及我们如何解释在颗粒组装过程中含有psi的RNA的选择性包装？_____我们发现，当Gag在没有vRNA的情况下在体内表达时，它几乎可以包装任何细胞mRNA。同样，在HIV-1 Gag中添加几乎任何核酸都会导致体外病毒样颗粒（VLP）组装。因此，vRNA在包装方面与mrna竞争；Psi包装信号使其在这场竞争中具有优势。我们正在利用荧光相关光谱研究重组Gag与含psi和控制rna的结合。我们发现Gag可以协同结合rna。Gag对Psi的亲和力仅略高于对照RNA。使用几个Gag突变体，我们发现与控制的结合主要归因于矩阵结构域。值得注意的是，与Psi结合具有不同的特性，因为它比与控制RNA结合具有更强的耐盐性，表明具有更高的非静电成分。我们正在测试这样一种想法，即与Psi结合会触发Gag经历的构象转变，使其比其他rna更有效地进入组装就绪状态。我们发现与Psi结合比与其他rna结合更有效地支持颗粒组装。这可以解释vRNA的选择性包装。数据还确定了Psi内的特定核苷酸，这些核苷酸是这种有效粒子组装所必需的。_____与该项目相关的专利：美国专利#5,674,720：“基因组RNA缺乏的非传染性人类逆转录病毒突变体的设计和构建”；1997年10月7日发布；Robert J. Gorelick, Larry O. Arthur, Alan Rein, Louis E. Henderson和Stephen Oroszlan。该专利描述了结构正常但非传染性的HIV-1突变体；这些突变体可能被视为疫苗成分。美国专利#7,572,828：“抑制病毒组装和核衣壳蛋白与核酸结合的抗hiv化合物的鉴定”；2009年8月11日发布；Robert Shoemaker, Michael Currens, Alan Rein, Ya Xiong Feng, Robert Fisher, Andrew Stephen, Shizuko Sei, Bruce crisis， Louis Henderson和Karen Worthy。这项专利描述了一类具有抗hiv -1活性的化合物，正在研究将其用于抗逆转录病毒治疗。专利申请：03773233.6 （EP申请）。

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. 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. Patent pending: 03773233.6 (EP application).