The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle
The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle
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DOI：
10.1128/jvi.03152-12
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发表时间：
2013-08-01
影响因子：
5.4
通讯作者：
Colombo, Maria I.
中科院分区：
文献类型：
--
作者：
Delgui, Laura R.;Rodriguez, Jose F.;Colombo, Maria I.
传染性法氏囊病病毒(IBDV)是一种双链RNA病毒，属于禽类病毒科，可引起鸡免疫抑制。在本研究中，我们基于对IBDV核糖核蛋白(RNPs)的主要蛋白质组分VP3的共定位分析，定义了IBDV复制复合体的定位。我们的结果表明，VP3定位于囊泡结构，具有位于核旁区域的早期和晚期内吞液室的特征。在内化步骤之后，用显性阴性版本的Rab5干扰内吞途径会导致病毒滴度的降低。VP3、病毒RNA依赖RNA聚合酶VP1和病毒双链RNA(DsRNA)之间的三重免疫染色研究表明，RNPs的三个关键成分共定位在同一结构中，可能代表复制复合体。有趣的是，重组表达的VP3也定位于内吞体内。利用高尔基体标记，我们发现含有VP3的囊泡与该细胞器密切相关。诺考达唑解聚微管后，VP3的定位发生了深刻的变化，呈点状分布于细胞质中。然而，这些VP3阳性结构仍然与高尔基氏肌有关。同样，灯盏花素A(BFA)处理导致VP3呈点状分布，散布在感染细胞的细胞质中。此外，对BFA处理禽类细胞后的细胞内和细胞外病毒感染颗粒的分析表明，高尔基复合体在病毒组装中发挥了作用。这些结果首次阐明了IBDV复制复合体的定位(即在胞内室)，并确立了高尔基体在禽流感病毒组装步骤中的作用。
Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra-and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.