Department of Physics, Northeastern University, Boston, MA 02115, USA.
Department of Chemistry and Biochemistry, The Ohio State University, Center for Retroviral Research, and Center for RNA Biology, Columbus, OH 43210, USA.
Viruses. 2020 Apr 25;12(5):484. doi: 10.3390/v12050484.
Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag) polyprotein, binds the genomic RNA and facilitates packaging into new virions. It is not clear how the same protein, alone or as part of Gag, performs such different RNA binding functions in the viral life cycle. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. Comparing measured results with a model of discrete protein binding allows us to localize affected binding sites, in addition to quantifying hairpin stability. We find that, while both NCp7 and GagDp6 destabilize the TAR hairpin, GagDp6 binding is localized to two sites in the stem, while NCp7 targets sites near the top loop. Unlike GagDp6, NCp7 destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by ~10. Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion.
逆转录病毒核衣壳（NC）蛋白是核酸伴侣，在病毒生命周期中发挥着不同的作用。在逆转录过程中，HIV-1 NC 促进核酸二级结构的重排，使 TAR RNA 发夹结构短暂失稳并与互补 RNA 发夹退火。相反，在病毒组装过程中，NC 作为群特异性抗原（Gag）多蛋白的一个结构域，结合基因组 RNA 并促进其包装到新的病毒粒子中。目前尚不清楚同一蛋白单独或作为 Gag 的一部分如何在病毒生命周期中执行如此不同的 RNA 结合功能。通过将单分子光学镊子测量与基于 mfold 的定量模型相结合，我们对 TAR RNA 的平衡稳定性和展开势垒进行了表征。将测量结果与离散蛋白结合模型进行比较，使我们能够定位受影响的结合位点，同时定量发夹稳定性。我们发现，尽管 NCp7 和 GagDp6 都使 TAR 发夹不稳定，但 GagDp6 结合定位于茎部的两个位点，而 NCp7 则靶向顶部环附近的位点。与 GagDp6 不同，NCp7 使该环不稳定，将反应势垒的位置向折叠状态移动，并使发夹打开的自然速率增加约 10 倍。因此，我们的结果解释了为什么 Gag 切割和 NC 释放是病毒内逆转录的必要前提。
