Generation of Mature DENVs via Genetic Modification and Directed Evolution.
Generation of Mature DENVs via Genetic Modification and Directed Evolution.
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作者：
登革热病毒（denv）的成熟改变了病毒粒子的结构、免疫力和传染性，高度成熟的颗粒在体内占主导地位。高度成熟病毒粒子的产生主要依赖于病毒早膜蛋白（prM）的结构和功能以及宿主蛋白酶furin对其的裂解。我们重新开发了一种可靠的克隆细胞系（VF1），它可以产生单轮成熟的DENV，而无需对DENV进行反向遗传。更重要的是，利用蛋白质工程和prM切割位点的定向进化，我们设计了在所有血清型中独立于细胞或宿主的遗传稳定的成熟denv，通常对病毒产量的影响最小。使用这些互补策略来调节成熟，我们证明了所产生的成熟denv在抗原性上不同于它们的等基因部分成熟形式。鉴于成熟DENV在免疫中的临床重要性，我们的研究为生产稳定、高滴度的成熟DENV提供了可靠的策略和试剂，用于DENV抗体中和和疫苗免疫研究。生物学上，我们从宿主物种的定向进化中获得的数据揭示了哺乳动物和昆虫细胞之间不同的成熟依赖选择压力，验证了哺乳动物和昆虫furin之间的底物偏好，同时暗示了DENV prM切割位点在宿主和载体之间的进化平衡。
Maturation of dengue viruses (DENVs) alters the structure, immunity, and infectivity of the virion and highly mature particles represent the dominant form in vivo. The production of highly mature virions principally relies on the structure and function of the viral premature membrane protein (prM) and its cleavage by the host protease furin. We redeveloped a reliable clonal cell line (VF1) which produces single-round mature DENVs without the need for DENV reverse genetics. More importantly, using protein engineering and directed evolution of the prM cleavage site, we engineered genetically stable mature DENVs in all serotypes independent of cell or host, usually with minimal impact on viral yield. Using these complementary strategies to regulate maturation, we demonstrate that the resulting mature DENVs are antigenically distinct from their isogenic partially mature forms. Given the clinical importance of mature DENVs in immunity, our study provides reliable strategies and reagents for the production of stable, high-titer mature DENVs for DENV antibody neutralization and vaccination immunity studies. Biologically, our data from directed evolution across host species reveals distinct maturation-dependent selective pressures between mammalian and insect cells, verifying the substrate preference between mammalian and insect furin, while hinting at an evolutionary equilibrium of DENV prM cleavage site between its host and vector in nature.
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影响因子：
13.6
作者：
Newton ND;Hardy JM;Modhiran N;Hugo LE;Amarilla AA;Bibby S;Venugopal H;Harrison JJ;Traves RJ;Hall RA;Hobson-Peters J;Coulibaly F;Watterson D
通讯作者：
Watterson D
影响因子：
16.6
作者：
Hardy JM;Newton ND;Modhiran N;Scott CAP;Venugopal H;Vet LJ;Young PR;Hall RA;Hobson-Peters J;Coulibaly F;Watterson D
通讯作者：
Watterson D
影响因子：
5.5
作者：
通讯作者：
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影响因子：
8.3
作者：
Ebi, Kristie L.;Nealon, Joshua
通讯作者：
Nealon, Joshua
影响因子：
5.4
作者：
Junjhon, Jiraphan;Edwards, Thomas J.;Sittisombut, Nopporn
通讯作者：
Sittisombut, Nopporn