Interactions of Retroviral Proteins with Nucleic Acids

逆转录病毒蛋白质和核酸之间似乎有几种不同的相互作用模式，每一种模式都对病毒复制具有重要的功能后果。首先，在病毒组装过程中，Gag多蛋白(病毒颗粒的结构蛋白)通过精妙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，正如我们的逆转录病毒组装和成熟项目(Zia BC 010511)所讨论的那样，病毒RNA所起的作用之一是使Gag蛋白紧密结合在一起。同时，成功的复制需要选择含有包装信号(称为Psi)的病毒基因组RNA(VRNA)进行包装，而不是选择数千种也是潜在包装底物的细胞RNA物种进行包装。最后，HIV-1 Gag及其裂解产物核衣壳蛋白(Nc)都具有核酸伴侣活性，使它们能够催化核酸重排到最低自由能的构象。似乎GAG中的这一活性负责将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。为了更好地了解HIV-1RNA包装和组装之间的关系，我们正在解决以下问题：HIV-1Gag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，它可以包装几乎任何细胞的mRNA。同样，在HIV-1Gag中加入几乎任何核酸都会在体外导致病毒样颗粒(VLP)的组装。因此，vRNA在包装方面与mRNAs竞争；Psi包装信号使其在这场竞争中具有优势。我们正在使用荧光相关光谱、微尺度热电泳法、SwitchSense仪器和质量光度法研究重组GAG与含有Psi的RNA和对照RNA的结合。我们发现GAG与RNA协同结合。GAG对Psi的亲和力仅略高于对照RNA。使用几个Gag突变体，我们发现与对照的结合在很大程度上归因于基质结构域。值得注意的是，与Psi的结合具有不同的特征，因为它比与控制RNA的结合更耐盐，表明有更高的非静电成分。我们正在测试这样一种想法，即与Psi的结合触发了GAG比其他RNA更有效地进入组装就绪状态的构象转变。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。这就解释了vRNA的选择性包装。这些数据还确定了Psi中的特定核苷酸，这些核苷酸是这种有效的粒子组装所必需的。与该项目相关的_项专利：美国专利#5,674,720：“缺乏基因组RNA的非传染性人类逆转录病毒突变体的设计和构建”；颁发于1997年10月7日；Robert J.Gorelick，Larry O.Arthur，Alan Rein，Louis E.Henderson和Stephen Oroszlan。这项专利描述了结构正常但不具传染性的HIV-1突变体；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核衣壳蛋白与核酸的结合的抗艾滋病毒化合物”；2009年8月11日颁发；Robert Shoemaker、Michael Currens、Alan Rein、冯亚雄、Robert Fisher、Andrew Stephen、Shizuko Sei、Bruce Crise、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，正在研究中，用于抗逆转录病毒治疗。

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.