Role of Us9 phosphorylation in axonal sorting and anterograde transport of pseudorabies virus.
Role of Us9 phosphorylation in axonal sorting and anterograde transport of pseudorabies virus.
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DOI：
10.1371/journal.pone.0058776
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Enquist LW
中科院分区：
文献类型：
--
作者：
Kratchmarov R;Taylor MP;Enquist LW
α疱疹病毒，如伪狂犬病病毒（PRV），在神经元轴突中经历顺行运输以促进宿主内的顺行传播。病毒粒子的轴突分选和顺行运输依赖于病毒膜蛋白Us 9，其与宿主马达蛋白Kif 1A相互作用以指导运输。Us 9-Kif 1A相互作用对于这些过程是必要的，但不是充分的，这表明需要额外的辅因子或翻译后修饰。在这项研究中，我们的特点是两个保守的丝氨酸磷酸化位点（S51和S53）的PRV Us 9蛋白是必要的顺行传播在体内。我们评估了亚细胞定位的磷酸-Us 9亚种在感染的神经元，发现磷酸形式是检测到的大多数，但不是全部，轴突囊泡含有Us 9蛋白。在生物化学测定中，磷酸-Us 9富集在脂筏膜微区，虽然Us 9磷酸化不需要事先脂筏协会。在感染的分室神经元培养物，我们观察到只有适度的减少，顺行传播能力的dispersant突变体Us 9，没有缺陷的单丝氨酸突变体。相反，邻近磷酸化位点的激酶识别序列残基的突变完全废除了顺行传播。在活细胞成像分析中，病毒体的顺行运输在用表达GFP-标记的discreteutrophin突变体Us 9的重组PRV株感染期间减少。Us 9-Kif 1A相互作用不需要磷酸化，这表明Us 9-Kif 1A结合是与转运复合物的激活和/或稳定不同的步骤。总之，我们的研究结果表明，虽然不是必需的，但Us 9磷酸化增强了轴突中基于Us 9-Kif 1A的病毒体转运，以调节感染长距离顺行传播的整体效率。
Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.
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DOI：
10.1083/jcb.200011146
发表时间：
2001-08-20
期刊：
The Journal of cell biology
影响因子：
--
作者：
Tomishima MJ;Enquist LW
通讯作者：
Enquist LW
影响因子：
--
作者：
Curanovic, Dusica;Ch'ng, Toh Hean;Szpara, Moriah;Enquist, Lynn
通讯作者：
Enquist, Lynn
影响因子：
11.1
作者：
Kratchmarov, R.;Taylor, M. P.;Enquist, L. W.
通讯作者：
Enquist, L. W.
影响因子：
30.3
作者：
Kramer T;Greco TM;Taylor MP;Ambrosini AE;Cristea IM;Enquist LW
通讯作者：
Enquist LW
影响因子：
3.7
作者：
Cohen, JI;Sato, H;Lekstrom, K
通讯作者：
Lekstrom, K