Interactions of Retroviral Proteins with Nucleic Acids

所有正常的逆转录病毒颗粒都含有基因组RNA的二聚体。二聚体的物理结构以及两个单体之间的连接尚不清楚。几年前，我们报道了病毒核衣壳蛋白在病毒成熟过程中从Gag中释放出来时改变了单体之间的连接。这种二聚体连接的稳定性是由核衣壳的核酸伴侣活性引起的。我们继续探索真实病毒颗粒内病毒RNA的结构。我们最近分析了RNA，通过使用RNase H和与RNA中的特定序列互补的短寡脱氧核苷酸在特定位点切割RNA。这些实验的结果首次将未成熟和成熟鼠白血病病毒（MLV）颗粒中单体之间最稳定的连接定位于其5'端附近的区域。实验还揭示了单体之间存在额外的、不太稳定的键。逆转录病毒核衣壳蛋白是高活性的核酸分子伴侣。这一关键活动的机制尚不清楚。我们已经分析了HIV-1核衣壳的结合非常短（8碱基）的寡脱氧核苷酸的非常详细，使用几种生物物理技术。我们发现单个核衣壳分子可以同时结合两个核酸分子;相反，单个核酸分子可以结合两个核衣壳分子。似乎蛋白质与一个以上核酸分子相互作用的能力是其核酸伴侣活性的关键要素。我们目前正在分析包装信号在HIV-1和MLV病毒RNA，使用遗传和结构的方法。最近的成就和目前的研究：A。γ逆转录病毒5'非翻译区的遗传分析在Kevin Weeks和Robert Gorelick博士的合作下，我们以前所未有的细节定义了MLV RNA的二聚化/包装信号。我们将进行进一步的致突变分析，并对其他γ逆转录病毒进行比较研究。我们还将对HIV-1 RNA的包装信号进行致突变研究。B. MLV Gag蛋白核酸伴侣活性的表征与Karin Musier-Forsyth博士合作，我们发现MA结构域可以干扰HIV-1 Gag的退火活性。我们将测试这是否也是真实的MLV Gag，尽管后者的扩展构象。C.细胞mRNA选择性包装的分析一些细胞mRNA在逆转录病毒颗粒中大量富集。我们将尝试识别其中之一的“包装信号”，即ASB-1 mRNA，并确定它是否以二聚体形式包装。D. MLV和HIV-1二聚体的结构研究与王云兴博士合作，我们将主要依靠小角X射线散射来研究MLV和HIV-1二聚体RNA的三维结构。e. HIV-1 Gag与核酸结合的溶液研究迄今为止，Gag蛋白与核酸的结合特征非常差。我们将在高离子强度下进行亲和力的基本测量，希望在这些条件下结合反应将被简化。与项目相关的专利：美国专利#5，674，720：“Design and Construction of Noninfectious Human Retroviral Mutants Deficient in Genomic RNA”; 1997年10月7日授权; Robert J. Gorelick，Larry O.放大图片作者：亚瑟，艾伦.亨德森和斯蒂芬·奥罗斯兰。该专利描述了HIV-1的突变体，这些突变体在结构上正常但不具有感染性;这些突变体可能被认为是疫苗成分。[对应于2011年11月艾滋病毒耐药性项目现场访问报告中的Rein项目2]

All normal retroviral particles contain a dimer of genomic RNA. The physical structure of the dimer, and of the linkage between the two monomers, is not understood. We reported some years ago that the viral nucleocapsid protein alters the linkage between the monomers when it is released from Gag during viral maturation. This stabilization of the dimeric linkage results from the nucleic acid chaperone activity of nucleocapsid. We have continued to probe the structure of viral RNA within authentic virus particles. We have recently analyzed the RNA by cleaving it at specific sites using RNase H and short oligodeoxynucleotides complementary to specific sequences in the RNA. The results of these experiments, for the first time, localized the most stable linkage between the monomers to the region near their 5' ends, in both immature and mature murine leukemia virus (MLV) particles. The experiments also revealed the existence of additional, less stable linkages between the monomers. Retroviral nucleocapsid proteins are highly active nucleic acid chaperones. The mechanism of this crucial activity is not well understood. We have analyzed the binding of HIV-1 nucleocapsid to a very short (8-base) oligodeoxynucleotide in great detail, using several biophysical techniques. We found that a single nucleocapsid molecule can bind simultaneously to two nucleic acid molecules; conversely, a single nucleic acid molecule can bind two nucleocapsid molecules. It seems likely that the ability of the protein to interact with more than one nucleic acid molecule is a critical element in its nucleic acid chaperone activity. We are currently analyzing packaging signals in both HIV-1 and MLV viral RNAs, using both genetic and structural methods. Recent Accomplishments and Current Research: a. Genetic analysis of gammaretroviral 5' untranslated regions In collaboration with Drs. Kevin Weeks and Robert Gorelick, we have defined the dimerization/packaging signal of MLV RNA in unprecedented detail. We will perform further mutagenic analysis and do comparative studies on other gammaretroviruses. We will also perform mutagenic studies on the packaging signal of HIV-1 RNA. b. Characterization of nucleic acid chaperone activity of MLV Gag protein In collaboration with Dr. Karin Musier-Forsyth, we have found that the MA domain can interfere with the annealing activity of HIV-1 Gag. We will test whether this is also true of MLV Gag, despite the extended conformation of the latter. c. Analysis of selective packaging of cellular mRNAs A few cellular mRNAs are greatly enriched in retrovirus particles. We will try to identify the "packaging signal" in one of them, viz. ASB-1 mRNA, and to determine whether it is packaged in dimeric form. d. Structural studies on dimers in MLV and HIV-1 In collaboration with Dr. Yun-Xing Wang, we will study the three-dimensional structure of dimeric RNAs of MLV and HIV-1, relying heavily on small-angle X-ray scattering. e. Solution studies of binding of HIV-1 Gag to nucleic acids The binding of Gag proteins to nucleic acids is very poorly characterized to date. We will make basic measurements of affinity at high ionic strength, in hopes that the binding reactions will be simplified under these conditions. Patent Linked to 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. [Corresponds to Rein Project 2 in the November 2011 site visit report of the HIV Drug Resistance Program]