Biochemical analysis of packing and assembling heptad repeat motifs in the coronavirus spike protein trimer.

IF 5.1 1区 生物学 Q1 MICROBIOLOGY mBio Pub Date : 2024-11-13 Epub Date: 2024-10-23 DOI:10.1128/mbio.01203-24
Jun Kobayashi, Kazuhiko Kanou, Hiyori Okura, Tahmina Mst Akter, Shuetsu Fukushi, Shutoku Matsuyama
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Abstract

During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology.

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冠状病毒穗状病毒三聚体中包装和组装七联重复图案的生化分析。
在冠状病毒感染过程中,尖峰蛋白在病毒和细胞膜界面上经历由其受体和宿主蛋白酶引发的连续结构转换,从而介导膜融合。受体结合后,病毒尖峰蛋白内的七重重复基序(HR1/HR2)将病毒膜和细胞膜连接起来;然而,由于尖峰蛋白的瞬时性和不稳定性,它将病毒膜和细胞膜拉近时所采用的中间构象仍不清楚。在这里,我们通过实验诱导了小鼠冠状病毒尖峰蛋白的构象变化,方法是将病毒与其受体孵育,然后暴露于胰蛋白酶。然后,我们用蛋白酶 K 处理病毒/受体复合物,以探究尖峰蛋白的紧密核心结构。尖峰蛋白的构象是通过蛋白酶消化产物的大小来预测的。受体结合后,检测到两条带(每条带与融合抑制 HR2 肽的反应性不同);我们认为这两条带对应于包装和未包装的 HR1/HR2 基序。在胰蛋白酶介导的触发后,温度和时间依赖性的测量显示,剩余未包装的 HR1/HR2 基序的包装和三个 HR1 基序在三聚体中的组装几乎同时发生。因此,三聚体尖峰蛋白在与受体结合后采用不对称的未组装构象,然后在宿主蛋白酶的触发下直接组装成融合后形式。这项生化研究从机理上揭示了病毒融合蛋白之前未知的中间结构。重要意义在包膜病毒感染期间,受体结合会触发细胞膜与病毒包膜之间的融合,从而将病毒基因组传递到细胞质中。病毒尖峰蛋白介导膜融合,但这一过程的分子机制尚不清楚。这是因为使用结构生物学方法来跟踪不稳定的尖峰三聚体中诱导的瞬时构象变化具有挑战性。在这里,我们利用蛋白酶识别蛋白质表面细微差别的能力,检测了构象变化前后尖峰蛋白的结构差异。降解产物的大小差异通过 Western 印迹分析进行了分析。本文提出的解释构象变化的模型是一个可信的候选模型,它为病毒学领域的未决问题提供了宝贵的见解。
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来源期刊
mBio
mBio MICROBIOLOGY-
CiteScore
10.50
自引率
3.10%
发文量
762
审稿时长
1 months
期刊介绍: mBio® is ASM''s first broad-scope, online-only, open access journal. mBio offers streamlined review and publication of the best research in microbiology and allied fields.
期刊最新文献
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