Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.
Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.
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DOI：
10.1073/pnas.2307997121
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发表时间：
2024-01-23
影响因子：
11.1
通讯作者：
Mitchison TJ
中科院分区：
文献类型：
--
作者：
Yoo TY;Mitchison TJ
SARS冠状病毒开放阅读框6 （ORF6）蛋白阻断核胞质转运以逃避宿主免疫，但其确切机制一直存在争议。解决这个问题将有助于我们了解SARS的演变和发病机制。为了解决这个问题，我们在单个活细胞中定量测量了ORF6和信息突变体对核转运抑制的浓度依赖性。SARS-CoV-2 ORF6明显比SARS-CoV-1 ORF6更有效，主要是由于其C端更短。n端区域促进ORF6寡聚化，这是与核孔多价相互作用所必需的，可以被合成的寡聚化物取代。我们的方法可以适应于测量任何蛋白质的浓度依赖性，其效果可以在单个活细胞中评分。开放阅读框6 （ORF6）蛋白是严重急性呼吸综合征相关（SARS）冠状病毒所特有的，可抑制经典的核输入途径以拮抗宿主抗病毒反应。提出了几种替代模型来解释ORF6的抑制功能[H]。夏等，Cell rep 33, 108234 (2020)；L. Miorin et al., Proc. Natl。学会科学。美国117,28344 - 28354 (2020)；李建军，张建军，李建军，等。生物信息学研究进展[j]。为了区分这些模型并定量了解ORF6的功能，我们开发了一种方法来对单个活细胞中的ORF6浓度和功能效果进行评分。我们将单细胞中未标记ORF6表达水平的定量与基于光遗传学的核转运动力学测量相结合，使用的方法可以适用于测量任何未标记蛋白的浓度依赖性效应。我们发现SARS-CoV-2 ORF6在抑制核进出口方面的效力是SARS-CoV-1 ORF6的15倍左右，这是由于NUP98-RAE1结合所需的c端区域不同。n端区域是运输抑制所必需的。这个区域与膜结合，但可以被人工合成的结构体取代，这些结构体在溶液中强制寡聚，这表明它的主要功能是寡聚。我们提出疏水的n端区域驱动ORF6的寡聚化，在核孔复合物上多价交联NUP98-RAE1复合物，这种多价结合抑制了双向运输。
SARS coronavirus Open Reading Frame 6 (ORF6) proteins block nucleocytoplasmic transport to evade host immunity, but the precise mechanism has been controversial. Solving this problem will help us understand SARS evolution and pathogenesis. To address this question, we made quantitative measurements of the concentration dependence of nuclear transport inhibition by ORF6, and informative mutants, in single living cells. SARS-CoV-2 ORF6 was strikingly more potent than SARS-CoV-1 ORF6, mainly due to its shorter C terminus. The N-terminal region promoted ORF6 oligomerization which was required for multivalent interaction with nuclear pores and could be replaced by synthetic oligomerizers. Our methods could be adapted to measure the concentration dependence of any protein whose effect can be scored in single live cells. Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al., Cell Rep. 33, 108234 (2020); L. Miorin et al., Proc. Natl. Acad. Sci. U.S.A. 117, 28344–28354 (2020); and M. Frieman et al., J. Virol. 81, 9812–9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98–RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98–RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.
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影响因子：
64.8
作者：
Ahn JH;Davis ES;Daugird TA;Zhao S;Quiroga IY;Uryu H;Li J;Storey AJ;Tsai YH;Keeley DP;Mackintosh SG;Edmondson RD;Byrum SD;Cai L;Tackett AJ;Zheng D;Legant WR;Phanstiel DH;Wang GG
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DOI：
10.1073/pnas.1716305114
发表时间：
2017-12-26
影响因子：
11.1
作者：
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影响因子：
8.8
作者：
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影响因子：
3.7
作者：
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影响因子：
6.4
作者：
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Greninger AL