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冠状病毒与种间膜蛋白和核衣壳蛋白嵌合体组装相互作用的分析

Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras.

作者信息

Kuo Lili, Hurst-Hess Kelley R, Koetzner Cheri A, Masters Paul S

机构信息

Wadsworth Center, New York State Department of Health, Albany, New York, USA.

Wadsworth Center, New York State Department of Health, Albany, New York, USA

出版信息

J Virol. 2016 Apr 14;90(9):4357-4368. doi: 10.1128/JVI.03212-15. Print 2016 May.

Abstract

UNLABELLED

The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought.

IMPORTANCE

The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.

摘要

未标记

冠状病毒膜(M)蛋白是病毒体形态发生的核心参与者。M蛋白组织病毒膜的各个组分,并且M蛋白自身之间以及与核衣壳(N)蛋白之间的相互作用驱动病毒组装和出芽。为了进一步明确M-M和M-N相互作用,我们构建了模型冠状病毒小鼠肝炎病毒(MHV)的突变体,其中全部或部分M蛋白被其来自严重急性呼吸综合征冠状病毒(SARS-CoV)的系统发育上不同的对应物所取代。我们能够获得含有完整SARS-CoV M蛋白的有活力的嵌合体以及具有分子内替换的突变体,这些突变体在胞外结构域、跨膜结构域或胞内结构域之间的边界处对M蛋白进行了划分。我们的结果表明,N蛋白的羧基末端结构域N3对于与M蛋白的相互作用是必要且充分的。然而,尽管先前有一些遗传和生化证据将与N的相互作用定位到M的羧基末端,但无法确定M蛋白中足以与N组装的短线性区域。因此,与N蛋白的相互作用可能涉及M胞内结构域的多个线性不连续区域。SARS-CoV M嵌合体表现出一种条件性生长缺陷,该缺陷被包膜(E)蛋白中的突变部分抑制。此外,M嵌合体的病毒体在刺突(S)蛋白掺入方面明显不足。这些发现表明,M蛋白与E蛋白和S蛋白的相互作用比先前认为的更为复杂。

重要性

冠状病毒病毒体的组装需要病毒结构蛋白和RNA基因组之间的协同相互作用。研究这一过程的一种策略是通过构建保留或破坏特定分子间或分子内关联的种间嵌合体。在这项工作中,我们用来自异源冠状病毒的对应物取代了模型冠状病毒小鼠肝炎病毒的膜(M)蛋白。结果阐明了我们对冠状病毒M蛋白与核衣壳蛋白之间相互作用的理解。同时,它们揭示了M与病毒刺突蛋白和包膜蛋白相互作用中意想不到的复杂性。

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