IMPORTANCE OF CONSERVED AMINO-ACIDS AT THE CLEAVAGE SITE OF THE HEMAGGLUTININ OF A VIRULENT AVIAN INFLUENZA-A VIRUS
IMPORTANCE OF CONSERVED AMINO-ACIDS AT THE CLEAVAGE SITE OF THE HEMAGGLUTININ OF A VIRULENT AVIAN INFLUENZA-A VIRUS
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DOI：
10.1099/0022-1317-74-2-311
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发表时间：
1993-02-01
影响因子：
3.8
通讯作者：
KAWAOKA, Y
中科院分区：
文献类型：
--
作者：
WALKER, JA;KAWAOKA, Y
甲型禽流感病毒的毒力取决于细胞内蛋白酶对多个碱性氨基酸的血凝素 (HA) 的裂解能力。尽管以前的研究已经证明了这些氨基酸对于细胞蛋白酶加工的重要性，重点是切割位点附近的保守残基，但切割的最低要求仍然未知。通过在猿猴病毒 40 系统中表达强毒力禽流感病毒 A/turkey/Ireland/1378/85 (H5N8) 的 HA 的位点特异性突变体，并测试它们在 CV-1 细胞中被内源蛋白酶切割的能力，以及它们在多核形成测定中的融合活性，我们能够证明 HA2 氨基末端的甘氨酸对于切割来说不是必需的，并且最大当碳水化合物在附近时，裂解需要在裂解位点至少有五个碱性残基。此外，我们证实，裂解位点上游的保守脯氨酸对于 HA 裂解或融合活性并不是必需的，并且 HA1 的羧基末端精氨酸的赖氨酸替换消除了可裂解性。这些发现应有助于确定负责细胞内裂解强毒禽流感病毒 HA 的蛋白酶。
The virulence of avian influenza A viruses depends on the cleavability of the haemagglutinin (HA) by an intracellular protease at multiple basic amino acids. Although previous studies have demonstrated the importance of these amino acids for processing by the cellular protease, with emphasis on conserved residues near the cleavage site, the minimal requirements for cleavage remain unknown. By expressing site-specific mutants of the HA of a virulent avian influenza virus, A/turkey/Ireland/1378/85 (H5N8), in the simian virus 40 system and testing for their cleavability by an endogenous protease in CV-1 cells, and their fusion activity in a polykaryon formation assay, we were able to show that glycine at the amino terminus of HA2 is not essential for cleavage and that maximal cleavage requires at least five basic residues at the cleavage site, when carbohydrate is nearby. Moreover, we confirmed, that a conserved proline upstream of the cleavage site is not essential for HA cleavage or fusion activity, and that lysine replacement of the carboxyl-terminal arginine of HA1 abolishes cleavability. These findings should help identify the proteases responsible for intracellular cleavage of the HA of virulent avian influenza viruses.