Ali A, Avalos R T, Ponimaskin E, Nayak D P
Department of Microbiology, Immunology and Molecular Genetics, Molecular Biology Institute, Johnsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90095-1747, USA.
J Virol. 2000 Sep;74(18):8709-19. doi: 10.1128/jvi.74.18.8709-8719.2000.
Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.
流感病毒基质蛋白（M1）是病毒组装和出芽所必需的关键蛋白，推测其在外层与病毒糖蛋白相互作用，在内层与病毒核糖核蛋白相互作用。然而，由于M1蛋白固有的膜结合能力，一直难以证明M1蛋白与流感病毒包膜糖蛋白血凝素（HA）或神经氨酸酶（NA）之间的特异性相互作用。通过用Triton X-100（TX-100）去污剂处理膜组分并在蔗糖梯度中进行漂浮实验，我们观察到单独表达或与异源仙台病毒F共表达的膜结合M1蛋白完全可溶于TX-100，但在HA和NA存在下表达的膜结合M1蛋白主要抗去污剂，并漂浮到密度梯度的顶部。此外，HA的细胞质尾和跨膜结构域都促进了M1与抗去污剂膜的结合。对流感病毒感染周期早期和晚期M1的膜结合分析表明，M1与成熟糖蛋白的相互作用与抗去污剂脂筏相关，这是膜结合M1抗去污剂的原因。共聚焦显微镜免疫荧光分析也表明，在流感病毒感染的细胞中，一部分M1蛋白与HA共定位，并在通过胞吐途径转运到质膜的过程中与HA相关联。当M1和HA共表达且HA转运被莫能菌素处理阻断时，也获得了类似的共定位结果。这些研究表明，HA和NA都与流感病毒M1相互作用，并且HA通过其细胞质尾和跨膜结构域与M1结合。
