Ly H, Nierlich D P, Olsen J C, Kaplan A H
Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, North Carolina, USA.
J Virol. 2000 Nov;74(21):9937-45. doi: 10.1128/jvi.74.21.9937-9945.2000.
Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255-7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.
已鉴定出几种似乎能促进鼠逆转录病毒基因组RNA二聚化的决定因素。这些决定因素之间的相互作用尚未得到广泛研究。此前，我们提出莫洛尼鼠肉瘤病毒基因组RNA的二聚化依赖于一个保守回文序列的浓度依赖性相互作用，该相互作用由单独的富含G的序列引发（H. Ly、D. P. Nierlich、J. C. Olsen和A. H. Kaplan，《病毒学杂志》73:7255 - 7261，1999）。使用遗传和反义方法相结合的方式研究了这两个元件的协同作用。携带回文序列和富含G序列的RNA分子的二聚化被与回文序列互补的寡核苷酸完全抑制；缺乏回文序列的分子在与两个富含G序列杂交的寡聚物存在下不能二聚化。自发二聚化实验的结果还表明，缺乏两段鸟嘌呤序列中任何一段的RNA分子二聚化速度比包含二聚体连接结构（DLS）的全长分子慢得多。然而，添加与剩余鸟嘌呤序列互补的寡核苷酸可将二聚化动力学恢复到野生型水平。这种寡聚物挽救二聚化动力学的能力取决于回文序列的存在，这表明富含G区域内的相互作用会使回文序列发生变化，从而使二聚化以最大效率进行。此外，在缺乏这些元件各种组合的构建体之间产生异源二聚体的尝试未成功，这表明富含G区域和回文序列不直接相互作用。最后，我们证明这两个元件对于维持有效的病毒复制都很重要。发现靶向DLS的修饰反义寡核苷酸会降低病毒载体滴度的产生水平。病毒滴度的降低是由于病毒基因组RNA包装效率的降低。总体而言，我们的数据支持一种逆转录病毒RNA二聚化的动态模型，其中离散的二聚化元件以协同方式起作用。
