Sequence dependencies and biophysical features both govern cleavage of diverse cut-sites by HIV protease.
Sequence dependencies and biophysical features both govern cleavage of diverse cut-sites by HIV protease.
复制标题
序列依赖性和生物物理特征都控制着 HIV 蛋白酶对不同切割位点的切割。
DOI：
10.1002/pro.4366
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Bolon,DanielNA
中科院分区：
文献类型：
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作者：
Samant,Neha;Nachum,Gily;Tsepal,Tenzin;Bolon,DanielNA
HIV-1的感染性需要其蛋白酶（PR）切割多个具有低序列相似性的切割位点。切割位点的多样性使得研究决定PR底物结合和周转的潜在序列特性具有挑战性。我们设计了一种突变扫描方法，利用酵母展示，流式细胞术和深度测序系统地测量三个不同切割位点（MA/CA，NC/p1和p1/p6）12个位置上所有单个氨基酸变化的影响。由此产生的适应性景观揭示了共同的物理特征，这些物理特征是在最接近易断裂键的氨基酸位置处切割所有三个切割位点的基础。相比之下，距离易断裂键超过两个氨基酸的位置表现出对切割位点其余部分的序列背景的强烈依赖性。我们观察到切割位点中的多个氨基酸变化导致更快的切割速率，包括在蛋白酶表面带正电荷的位置处远离易断裂键的5个和6个氨基酸的负电荷偏好。使用全长基质衣壳蛋白对单个切割位点的分析表明，长距离序列环境可能有助于切割效率，因此应仔细考虑对肽或较短工程构建体（包括本工作中的构建体）的分析。这项工作为理解不同底物如何与HIV-1 PR相互作用提供了一个框架，并可以扩展到研究具有类似性质的其他病毒PR。
The infectivity of HIV‐1 requires its protease (PR) cleave multiple cut‐sites with low sequence similarity. The diversity of cleavage sites has made it challenging to investigate the underlying sequence properties that determine binding and turnover of substrates by PR. We engineered a mutational scanning approach utilizing yeast display, flow cytometry, and deep sequencing to systematically measure the impacts of all individual amino acid changes at 12 positions in three different cut‐sites (MA/CA, NC/p1, and p1/p6). The resulting fitness landscapes revealed common physical features that underlie cutting of all three cut‐sites at the amino acid positions closest to the scissile bond. In contrast, positions more than two amino acids away from the scissile bond exhibited a strong dependence on the sequence background of the rest of the cut‐site. We observed multiple amino acid changes in cut‐sites that led to faster cleavage rates, including a preference for negative charge five and six amino acids away from the scissile bond at locations where the surface of protease is positively charged. Analysis of individual cut sites using full‐length matrix‐capsid proteins indicate that long‐distance sequence context can contribute to cutting efficiency such that analyses of peptides or shorter engineered constructs including those in this work should be considered carefully. This work provides a framework for understanding how diverse substrates interact with HIV‐1 PR and can be extended to investigate other viral PRs with similar properties.