Full-length three-dimensional structure of the influenza A virus M1 protein and its organization into a matrix layer

Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein-protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy. The resulting 3.4 Å-resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses.

许多包膜病毒，包括流感病毒、疱疹病毒和冠状病毒，编码了基质蛋白。在流感病毒的病毒包膜下方，基质蛋白1（M1）形成了一个对颗粒稳定性和pH依赖性RNA基因组释放至关重要的寡聚层。然而，全长的单聚体M1及其基质层的解析结构尚未获得，这阻碍了抗病毒靶向和对细胞进入过程中pH依赖性转换的理解。在本研究中，通过纯化和广泛的突变研究，揭示了形成类似于在细胞进入时暴露于低pH环境的病毒颗粒中观察到的多层螺旋M1寡聚体所需的蛋白-蛋白界面。然而，在单个氨基酸突变后，观察到与细胞进入前在感染性病毒颗粒中发现的单层螺旋寡聚体在生化及超微结构上的相似性。单层寡聚体的高度有序结构和其与完整病毒颗粒基质层的相似性，促使我们通过冷冻电子显微镜对其进行结构分析。所得3.4埃分辨率的结构揭示了M1折叠的分子细节及其在单壳层基质中的组织。解决全长M1结构、识别关键组装界面以及开发由纯化蛋白组成的M1组装检测，对于流感病毒的抗病毒靶向研究具有重要的进展。