Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza A virus neuraminidase in raft association and virus budding
Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza A virus neuraminidase in raft association and virus budding
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DOI：
10.1128/jvi.78.10.5258-5269.2004
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发表时间：
2004-05-01
影响因子：
5.4
通讯作者：
Nayak, DP
中科院分区：
文献类型：
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作者：
Barman, S;Adhikary, L;Nayak, DP
流感病毒神经氨酸酶(NA)是一种II型跨膜糖蛋白，具有破坏受体的活性，从而促进病毒从细胞表面释放。在甲型流感病毒中，NA的细胞质尾部(CT)和跨膜区(TMD)氨基酸序列都高度保守，但它们在病毒生物学中的功能(S)尚不清楚。为了研究CT和TMD的氨基酸序列在病毒生命周期中的作用，我们通过将2到5个相邻的氨基酸转化为丙氨酸来系统地诱变NA的整个CT和TMD。此外，我们还用人转铁蛋白受体tr(一种II型跨膜糖蛋白)取代了NA TMD[NA(1T2N)NA]的CT近三分之一氨基酸和整个NA TMD(NatRNA)的CT近端1/3氨基酸，从而制成了两个嵌合NA。我们通过反向遗传学挽救了转染型突变病毒，并对其表型进行了检测。我们的结果表明，所有突变和嵌合的Nas都可以被拯救成转基因病毒。不同的突变体对病毒的生长和复制表现出多效性。一些突变体(NA2A5、NA3A7和NA4A10)对病毒的生长几乎没有影响，而另一些(NA3A2、NA5A27和NA5A31)产生的侵染性病毒约为原来的50-100倍，还有一些(NA5A14、NA4A19和NA4A23)表现出中间表型。总体而言，TMD胞外区-近端序列的突变逐渐导致NA酶活性降低，影响脂筏结合，并抑制病毒生长。电子显微镜分析表明，这些突变病毒仍然聚集并结合在感染细胞表面，并可通过细菌NA处理从感染细胞中释放出来。此外，含有CT(NA3A2)N端突变的病毒和含有TMD的嵌合NA用tr TMD部分取代了[NA(1T2N)NA]或完全(NatRNA)，导致病毒生长减少，并表现出细长颗粒的形态表型。这些结果表明，尽管NA、CT和TMD本身的序列对病毒的生命周期并不是绝对必要的，但特定的氨基酸序列在提供NA的结构稳定性、酶活性和脂筏结合方面起着关键作用。此外，一些突变病毒的异常形态发生，包括细长颗粒的形成，表明NA参与了病毒的形态发生和萌发。
Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.