Yadav Vibha, Panganiban Antonito T, Honer Zu Bentrup Kerstin, Voss Thomas G
Division of Microbiology, Tulane National Primate Research Center, Covington, LA USA.
Department of Microbiology and Immunology, Tulane School of Medicine, Tulane University, New Orleans, LA USA.
Virusdisease. 2016 Dec;27(4):357-368. doi: 10.1007/s13337-016-0347-3. Epub 2016 Sep 14.
Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi. Also, we carried out complementary studies to determine the effect of IFV on intracellular membranes. Inhibition of the CRM1 and TAP-P15 nuclear transport pathways by DRB and LB blocked completely the export of virus. Inhibition of vesicular trafficking by BFA, NOC, and ZWEHD also affected influenza infection. Interestingly, IFV infection induced fragmentation of the Golgi complex resulting in diffuse distribution of large and small vesicles throughout the cytoplasm. Live-cell microscopy revealed expansion of Golgi localization signals indicating progressive dispersion of Golgi positive structures, resulting in the disassembly of the Golgi ribbon structure. Other vesicular components (Rab1b, ARF1 and GBF1) were also found to be required for IFV infection. Furthermore, the exact step at which IFV infection disrupts vesicle trafficking was identified as the ER-Golgi intermediate compartment. These findings suggest that IFV NP is trafficked from the nucleus via the CRM1 and TAP pathways. IFV modulates vesicular trafficking inducing disruption of the Golgi complex. These studies provide insight on the ways in which IFV affects intracellular trafficking of different host proteins and will facilitate identification of useful pharmaceutical targets to abrogate virus replication.
甲型流感病毒（IFV）在受感染细胞的细胞核中复制其基因组，并利用细胞蛋白质转运系统将基因组从细胞核运输到质膜。然而，这一过程的机制细节及其与随后细胞质中病毒运输的关系尚未阐明。我们研究了核运输抑制剂雷帕霉素B（LB）、5,6-二氯-1-β-D-呋喃核糖基苯并咪唑（DRB）、囊泡运输抑制剂布雷菲德菌素A（BFA）、半胱天冬酶抑制剂ZWEHD和微管抑制剂诺考达唑（NOC）对病毒复制以及病毒核蛋白（NP）从细胞核到内质网和高尔基体的细胞内运输的影响。此外，我们还进行了补充研究以确定IFV对细胞内膜的影响。DRB和LB对CRM1和TAP-P15核运输途径的抑制完全阻断了病毒的输出。BFA、NOC和ZWEHD对囊泡运输的抑制也影响了流感感染。有趣的是，IFV感染导致高尔基体复合体碎片化，致使大小囊泡在整个细胞质中呈弥散分布。活细胞显微镜检查显示高尔基体定位信号扩展，表明高尔基体阳性结构逐渐分散导致高尔基体带状结构解体。还发现其他囊泡成分（Rab1b、ARF1和GBF1）也是IFV感染所必需的。此外，IFV感染破坏囊泡运输的确切步骤被确定为内质网-高尔基体中间区室。这些发现表明，IFV NP通过CRM1和TAP途径从细胞核运输。IFV调节囊泡运输，导致高尔基体复合体破坏。这些研究为IFV影响不同宿主蛋白细胞内运输的方式提供了见解，并将有助于确定消除病毒复制的有用药物靶点。
