Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread
Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread
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DOI：
10.1128/jvi.00036-16
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发表时间：
2016-05-01
影响因子：
5.4
通讯作者：
Fang, Ying
中科院分区：
文献类型：
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作者：
Guo, Rui;Katz, Benjamin B.;Fang, Ying
细胞间纳米管连接已被确定为某些病毒细胞传播的替代途径。在感染猪繁殖与呼吸综合征病毒（PRRSV）的细胞中，观察到纳米管连接两个遥远的细胞与相邻的膜，与核心感染性病毒机制（病毒RNA，某些复制酶，和某些结构蛋白）存在于/上的细胞间纳米管。活细胞电影追踪表达绿色荧光蛋白（GFP）标记的nsp 2的重组PRRSV的细胞间转运。在表达PRRSV受体的MARC-145细胞中，GFP-nsp 2在病毒中和抗体的存在下通过纳米管从一个细胞移动到另一个细胞。病毒蛋白质的细胞间转运不需要PRRSV受体，因为在用GFP-PRRSV的感染性克隆转染后在受体阴性的HEK-293 T细胞中观察到。此外，在与重组PRRSV感染的MARC-145细胞共培养的HEK-293 T细胞中检测到GFP-nsp 2。细胞间纳米管含有丝状肌动蛋白（F-肌动蛋白）与肌球蛋白相关的马达蛋白。F-肌动蛋白和肌球蛋白IIA被鉴定为与PRRSV nsp 1 β、nsp 2、nsp 2 TF、nsp 4、nsp 7-nsp 8、GP 5和N蛋白共沉淀。抑制肌动蛋白聚合或肌球蛋白IIA激活的药物阻止了病毒感染细胞中纳米管的形成和病毒簇。这些数据使我们提出，繁殖与呼吸综合征病毒利用纳米管内的宿主细胞细胞骨架机制进行有效的细胞间传播。这种形式的病毒运输代表了病毒传播的另一种途径，它对宿主的体液免疫反应有抵抗力。重要性细胞外病毒颗粒在生物体之间传播感染，但在受感染的宿主内，细胞间感染可以通过其他机制传播。在这项研究中，我们描述了一种替代途径的细胞间传播的PRRSV病毒使用纳米管连接运输传染性病毒RNA，某些复制酶，和某些结构蛋白到邻近细胞。这个过程涉及病毒蛋白与形成纳米管连接的细胞骨架蛋白的相互作用。通过纳米管的细胞间病毒传播允许病毒逃避中和抗体反应，并可能有助于病毒感染的发病机制。制定干预这一过程的策略对于防止病毒感染的传播至关重要。
Intercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two distant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain structural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV-infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor proteins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1 beta, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response.IMPORTANCEExtracellular virus particles transmit infection between organisms, but within infected hosts intercellular infection can be spread by additional mechanisms. In this study, we describe an alternative pathway for intercellular transmission of PRRSV in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases, and certain structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection.