Experimental Estimation of the Effects of All Amino-Acid Mutations to HIV's Envelope Protein on Viral Replication in Cell Culture.
Experimental Estimation of the Effects of All Amino-Acid Mutations to HIV's Envelope Protein on Viral Replication in Cell Culture.
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DOI：
10.1371/journal.ppat.1006114
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发表时间：
2016-12
期刊：
影响因子：
6.7
通讯作者：
Bloom JD
中科院分区：
文献类型：
--
作者：
Haddox HK;Dingens AS;Bloom JD
艾滋病毒因其通过快速序列进化逃避免疫和抗病毒药物的能力而臭名昭著。了解突变对HIV的功能影响对于理解这种进化至关重要。HIV进化最快的蛋白质是它的包膜（Env）。在这里，我们使用深度突变扫描来实验性地估计Env的所有氨基酸突变对细胞培养物中病毒复制的影响。在我们的实验中，大多数突变都处于纯化选择之下，尽管少数位点经历了增强HIV在细胞培养中复制的突变的强选择。我们比较我们的实验测量每个网站的偏好，每个氨基酸的实际频率，这些氨基酸在自然发生的HIV序列。我们测量的氨基酸偏好与大多数位点的天然序列中的氨基酸频率相关。然而，我们测量的偏好是不太一致的天然氨基酸频率在表面暴露的网站，受到压力缺席，从我们的实验，如抗体选择。我们的数据使我们能够量化Env中每个位点的固有突变耐受性。我们表明，广泛中和抗体的表位具有显著降低的固有耐受突变的能力，严格验证了该领域的普遍想法。总的来说，我们的研究结果有助于解开固有的功能限制和外部选择压力在塑造环境的演变的作用。HIV因其表面蛋白Env的快速进化而臭名昭著。在实验室中确定的选择压力下测量所有突变对Env的影响的能力将为更好地理解塑造这种进化的因素打开大门。然而，这是一项艰巨的实验任务，因为Env有超过104种不同的单氨基酸突变。在这里，我们利用下一代测序来进行一个大规模平行实验，估计所有这些突变对细胞培养中病毒复制的影响。我们的测量结果在很大程度上与现有的知识一致的突变的影响，在功能上重要的网站，并显示，固有的突变耐受性变化很大，跨环境。我们的工作为Env的进化提供了新的见解，并描述了一种强大的实验方法，用于测量突变对实验室中可选择的HIV表型的影响。
HIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV’s most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV’s replication in cell culture. We compare our experimental measurements of each site’s preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. Our data enable us to quantify the inherent mutational tolerance of each site in Env. We show that the epitopes of broadly neutralizing antibodies have a significantly reduced inherent capacity to tolerate mutations, rigorously validating a pervasive idea in the field. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env’s evolution. HIV is infamous for the rapid evolution of its surface protein, Env. The ability to measure the effects of all mutations to Env under defined selection pressures in the lab would open the door to better understanding the factors that shape this evolution. However, this is a daunting experimental task since there are over 104 different single-amino acid mutations to Env. Here we leverage next-generation sequencing to perform a single massively parallel experiment that estimates the effects of all these mutations on viral replication in cell culture. Our measurements are largely consistent with existing knowledge about the effects of mutations at functionally important sites, and show that inherent mutational tolerance varies widely across Env. Our work provides new insight into Env’s evolution, and describes a powerful experimental approach for measuring the effects of mutations on HIV phenotypes that can be selected for in the lab.
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影响因子：
32.4
作者：
Ferguson AL;Mann JK;Omarjee S;Ndung'u T;Walker BD;Chakraborty AK
通讯作者：
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影响因子：
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