HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Viruses. 2020 Apr 2;12(4):394. doi: 10.3390/v12040394.
Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag-RNA and Gag-Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.
病毒基因组 RNA 具有高度特异性和选择性地被包装到病毒粒子中。然而，在含有生理盐的缓冲液中测量时，Gag 对病毒 RNA 的特异性会被掩盖。有趣的是，当结合受到盐浓度增加、竞争 RNA 的添加或引入 Gag 蛋白突变的挑战时，对病毒 RNA 的特异性变得可检测。这项工作的目的是研究 HIV-1 Gag 多蛋白结构域对非特异性和特异性 RNA 结合以及初始蛋白-RNA 复合物稳定性的贡献。使用一组具有突变的 Gag 蛋白，这些突变使不同的 Gag-Gag 或 Gag-RNA 界面失活，我们通过测量蛋白质与 RNA 的结合来研究区分病毒和非病毒 RNA 结合的各个结构域的独特贡献。我们在接近生理盐浓度下测量结合亲和力，然后通过增加离子强度来挑战结合，以抑制静电相互作用并揭示特定 Gag-RNA 和 Gag-Gag 相互作用的贡献。令人惊讶的是，我们观察到 Gag 二聚化和基质结构域中的高度碱性区域对病毒 RNA 结合的特异性有很大贡献。
