Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
RNA. 2013 Aug;19(8):1078-88. doi: 10.1261/rna.038869.113. Epub 2013 Jun 24.
Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.
尽管细胞 RNA 大量过剩，但通过 HIV-1 Gag 和 gRNA psi（ψ）包装信号之间的特异性相互作用，恰好有两个拷贝的病毒基因组 RNA（gRNA）被选择性地包装到新的人类免疫缺陷病毒 1 型（HIV-1）颗粒中。Gag 由基质（MA）、衣壳、核衣壳（NC）和 p6 结构域组成。Gag NC 结构域与 ψ 的结合对于 gRNA 包装是必需的，但 Gag 如何选择性地与 ψ 相互作用的机制尚不清楚。在这里，我们研究了 NC 和 Gag 变体与来自 ψ（Psi RNA）的 RNA 以及非 ψ 区域（TARPolyA）的结合。结合作为盐的函数进行测量，以获得有效电荷（Zeff）和结合的非静电（即特异性）部分，Kd(1M)。Gag 与 Psi RNA 的结合具有显著降低的 Kd(1M)和相对于 TARPolyA 降低的 Zeff。与 WT Gag 相比，NC、GagΔMA 和 Gag 的二聚化突变体与 TARPolyA 的结合具有降低的 Zeff。涉及 Gag 的 NC 锌指模体的突变或 Psi RNA 的富含 G 的 NC 结合区域的变化会显著降低结合的非静电部分，导致 Zeff 增加。这些结果表明，Gag 与 gRNA 相互作用使用不同的结合模式；在 TARPolyA 的情况下，NC 和 MA 结构域都与 RNA 结合，而与 Psi RNA 的结合仅涉及 NC 结构域。综上所述，这些结果提出了一种选择性 gRNA 封装的新机制。
