Cell Cycle-independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection.
Cell Cycle-independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection.
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DOI：
10.1074/jbc.m117.776112
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发表时间：
2017-03-24
期刊：
影响因子：
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通讯作者：
Sanyal S
中科院分区：
文献类型：
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作者：
Fan Y;Mok CK;Chan MC;Zhang Y;Nal B;Kien F;Bruzzone R;Sanyal S
为了鉴定调节甲型流感病毒复制的新宿主因子，我们使用来自高致病性H5 N1毒株的基质蛋白2的胞质尾进行酵母双杂交筛选。筛选结果显示与细胞周期蛋白D3（细胞周期早期G1期的关键调节因子）的高评分相互作用。M2-细胞周期蛋白D3相互作用通过GST下拉验证，并在流感病毒A/WSN/33感染的细胞中重现。通过小干扰RNA敲低Ccnd 3显著增强细胞培养上清液中的病毒子代滴度。有趣的是，病毒产量的增加是由于细胞周期蛋白D3缺陷本身，而不仅仅是细胞周期失调的结果。Ccnd 3和Rb 1的联合敲除，挽救了细胞周期进入S期，未能使病毒生产正常化。感染甲型流感病毒引发细胞周期蛋白D3从细胞核到细胞质的重新分配，随后其蛋白酶体降解。当在HEK 293 T细胞中过表达时，细胞周期蛋白D3损害了M2与M1的结合，这对于后代病毒体的正确组装是必不可少的，进一步支持其作为推定的限制因子的作用。我们的研究描述了细胞周期蛋白D3作为一种新的相互作用的流感病毒M2蛋白的鉴定和表征。我们推测，竞争性抑制M1-M2相互作用的细胞周期蛋白D3损害感染性病毒粒子的形成，并导致减毒病毒的生产。此外，我们还提供了有关感染期间流感病毒与宿主细胞周期机制动态相互作用的机制见解。
To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into S phase, failed to normalize virus production. Infection by influenza A virus triggered redistribution of cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. When overexpressed in HEK 293T cells, cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.