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。我们在几年前报道过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源于核衣壳的核酸伴侣活性。我们继续在真实的病毒颗粒中探测病毒RNA的结构。我们最近通过使用RNase H和短的寡脱氧核苷酸与RNA中的特定序列互补在特定位点切割RNA来分析RNA。这些实验的结果首次在未成熟和成熟的小鼠白血病病毒（MLV）颗粒中，将单体之间最稳定的连接定位在其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度活跃的核酸伴侣蛋白。这一关键活动的机制尚不清楚。我们使用几种生物物理技术，非常详细地分析了HIV-1核衣壳与非常短的（8碱基）寡脱氧核苷酸的结合。我们发现单个核衣壳分子可以同时与两个核酸分子结合；相反，一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的核酸分子相互作用的能力是其核酸伴侣活性的关键因素。我们目前正在使用遗传和结构方法分析HIV-1和MLV病毒rna中的包装信号。最近的成就和当前的研究：a. γ -逆转录病毒5'非翻译区域的遗传分析。Kevin Weeks和Robert Gorelick，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诱变分析，并对其他病毒进行比较研究。我们还将对HIV-1 RNA的包装信号进行诱变研究。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适用于MLV Gag，尽管后者的延伸构象。c.细胞mrna的选择性包装分析一些细胞mrna在逆转录病毒颗粒中大量富集。我们将尝试识别其中一个“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我们将与王云兴博士合作，主要依靠小角度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蛋白与核酸结合的特征非常少。我们将在高离子强度下进行基本的亲和测量，希望在这些条件下简化结合反应。与项目相关的专利：美国专利#5,674,720：“设计和构建缺乏基因组RNA的非传染性人类逆转录病毒突变体”；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。[对应2011年11月HIV耐药项目实地考察报告中的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]