Shubsda M F, McPike M P, Goodisman J, Dabrowiak J C
Department of Chemistry, Center for Science and Technology, Syracuse University, New York 13224-4100, USA.
Biochemistry. 1999 Aug 3;38(31):10147-57. doi: 10.1021/bi990744t.
The genome of the human immunodeficiency virus (HIV) exists as a dimer of two identical RNA molecules hydrogen bonded to each other near their 5' ends. The dimer, known to be important for viral infectivity, is formed by two monomers interacting through a stem-loop structure called the dimer initiation site (DIS). An initially formed intermediate, the "kissing" dimer, is unstable and rearranges to the stable, duplex form. In this report we use nondenaturing polyacrylamide gel electrophoresis to measure the monomer-dimer equilibrium constant of three RNA sequences, 41-, 27-, and 19-mers, located in the DIS of the MAL isolate of HIV-1. Experiments in which the RNA was equilibrated at various temperatures before electrophoresis revealed that interconversion is rapid for all the sequences, so that they reach equilibrium in the loading well of the gel at 5 degrees C before they enter the gel proper. However, interconversion kinetics in the gel are slow, so autoradiographic spot intensities can be used to measure the amounts of monomer and dimer present when the sample entered the gel. After correction for the amount of RNA added with the radiolabel and dilution of samples in the loading well of the gel, dimerization equilibrium constants were calculated from spot intensities. The calculated values of the dimerization constant K at 5 degrees C were approximately 10(5), approximately 10(6), and approximately 10(8) M(-1) for the 41-, 27-, and 19-mers, respectively, in solutions of ionic strength, I, of about 100 mM. The decrease in K by three orders of magnitude between the 19-mer and 41-mer is due in part to the change in rotational entropy of rodlike molecules on dimerization and in part to the increased conformational entropy of the monomers. As expected, increased ionic strength increases the dimerization constant for all three RNAs. For the 41-mer, however, K has a maximum value at I approximately 140 mM. The origin of the decrease in K for higher I is unknown but it may be due to formation of species (perhaps higher order oligomers) that do not enter the gel. The 41-mer exists in two dimeric forms assigned to the kissing and duplex dimers. The ratio of kissing to duplex form at 5 degrees C is 0.48 +/- 0.22 at I = 113 mM and 0.91 +/- 0.35 at I = 183 mM. The observed decrease in K with RNA length suggests that the dimerization constant of the packaging region of HIV-1 is small, < approximately 10(5) M(-1), implying that the nucleocapsid protein is important in promoting dimerization in the capsid of the virus.
人类免疫缺陷病毒（HIV）的基因组以两个相同RNA分子的二聚体形式存在，它们在5'端附近通过氢键相互结合。已知该二聚体对病毒感染性很重要，它由两个单体通过一种称为二聚体起始位点（DIS）的茎环结构相互作用形成。最初形成的中间体“亲吻”二聚体不稳定，会重排为稳定的双链形式。在本报告中，我们使用非变性聚丙烯酰胺凝胶电泳来测量位于HIV-1 MAL分离株DIS中的三个RNA序列（41聚体、27聚体和19聚体）的单体-二聚体平衡常数。在电泳前将RNA在不同温度下平衡的实验表明，所有序列的相互转化都很快，以至于它们在5摄氏度时在凝胶加样孔中达到平衡，然后才进入凝胶本身。然而，凝胶中的相互转化动力学很慢，因此放射自显影片斑点强度可用于测量样品进入凝胶时存在的单体和二聚体的量。在对用放射性标记添加的RNA量以及凝胶加样孔中样品的稀释进行校正后，根据斑点强度计算二聚化平衡常数。在离子强度I约为100 mM的溶液中，5摄氏度时41聚体、27聚体和19聚体的二聚化常数K的计算值分别约为10^5、约为10^6和约为10^8 M^-1。19聚体和41聚体之间K下降三个数量级，部分原因是棒状分子二聚化时旋转熵的变化，部分原因是单体构象熵的增加。正如预期的那样，离子强度增加会提高所有三种RNA的二聚化常数。然而，对于41聚体，K在I约为140 mM时具有最大值。I较高时K下降的原因尚不清楚，但可能是由于形成了不进入凝胶的物种（可能是高阶寡聚体）。41聚体以两种二聚体形式存在，分别对应于“亲吻”二聚体和双链二聚体。在I = 113 mM时，5摄氏度下“亲吻”二聚体与双链二聚体的比例为0.48±0.22，在I = 183 mM时为0.91±0.35。观察到的K随RNA长度的下降表明，HIV-1包装区域的二聚化常数很小，<约10^5 M^-1，这意味着核衣壳蛋白在促进病毒衣壳中的二聚化方面很重要。
