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二聚体。二聚体的物理结构以及两个单体之间的连接还不清楚。我们几年前就报道过，病毒核衣壳蛋白在病毒成熟过程中从GAG中释放时会改变单体之间的连接。这种二聚键的稳定是由于核衣壳的核酸伴侣活性所致。我们继续探索正宗病毒颗粒内的病毒RNA结构。我们最近用RNaseH和与RNA中特定序列互补的短寡核苷酸在特定位点切割RNA来分析RNA。这些实验的结果第一次在未成熟和成熟的小鼠白血病病毒颗粒中定位了单体之间最稳定的连接到其5‘末端附近的区域。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一种高活性的核酸伴侣蛋白。这一关键活动的机制还没有被很好地理解。我们使用几种生物物理技术，非常详细地分析了HIV-1核衣壳与一个非常短的(8个碱基)寡核苷酸的结合。我们发现，一个核衣壳分子可以同时与两个核酸分子结合；反之，一个核酸分子可以与两个核衣壳分子结合。蛋白质与多个核酸分子相互作用的能力似乎是其核酸伴侣活性的关键因素。[对应于2007年4月艾滋病毒耐药计划现场访问报告中的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 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. [Corresponds to Rein Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]