Méndez Fernando, Romero Nicolás, Cubas Liliana L, Delgui Laura R, Rodríguez Dolores, Rodríguez José F
Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología-CSIC, Madrid, Spain.
Instituto de Histología y Embriología de Mendoza - CONICET, Universidad Nacional de Cuyo, Mendoza, Argentina.
PLoS One. 2017 Jan 17;12(1):e0170080. doi: 10.1371/journal.pone.0170080. eCollection 2017.
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is responsible for a devastating immunosuppressive disease affecting juvenile domestic chickens. IBDV particles are naked icosahedrons enclosing a bipartite double-stranded RNA genome harboring three open reading frames (ORF). One of these ORFs codes for VP5, a non-structural polypeptide dispensable for virus replication in tissue culture but essential for IBDV pathogenesis. Using two previously described recombinant viruses, whose genomes differ in a single nucleotide, expressing or not the VP5 polypeptide, we have analyzed the role of this polypeptide during the IBDV replication process. Here, we show that VP5 is not involved in house-keeping steps of the virus replication cycle; i.e. genome transcription/replication, protein translation and virus assembly. Although infection with the VP5 expressing and non-expressing viruses rendered similar intracellular infective progeny yields, striking differences were detected on the ability of their progenies to exiting infected cells. Experimental data shows that the bulk of the VP5-expressing virus progeny efficiently egresses infected cells during the early phase of the infection, when viral metabolism is peaking and virus-induced cell death rates are as yet minimal, as determined by qPCR, radioactive protein labeling and quantitative real-time cell death analyses. In contrast, the release of the VP5-deficient virus progeny is significantly abridged and associated to cell death. Taken together, data presented in this report show that IBDV uses a previously undescribed VP5-dependent non-lytic egress mechanism significantly enhancing the virus dissemination speed. Ultrastructural analyses revealed that newly assembled IBDV virions associate to a vesicular network apparently facilitating their trafficking from virus assembly factories to the extracellular milieu, and that this association requires the expression of the VP5 polypeptide.
传染性法氏囊病病毒（IBDV）是双RNA病毒科的成员，可引发一种影响幼年家鸡的毁灭性免疫抑制疾病。IBDV颗粒为无包膜二十面体，包裹着一个二分体双链RNA基因组，该基因组含有三个开放阅读框（ORF）。其中一个ORF编码VP5，这是一种非结构多肽，在组织培养中对病毒复制并非必需，但对IBDV的发病机制至关重要。我们使用两种先前描述的重组病毒（其基因组仅在一个核苷酸上存在差异，分别表达或不表达VP5多肽），分析了该多肽在IBDV复制过程中的作用。在此，我们表明VP5不参与病毒复制周期的常规步骤，即基因组转录/复制、蛋白质翻译和病毒组装。尽管感染表达VP5和不表达VP5的病毒产生的细胞内感染性子代产量相似，但在它们的子代离开感染细胞的能力方面检测到了显著差异。实验数据表明，在感染早期，当病毒代谢达到峰值且病毒诱导的细胞死亡率尚未达到最低时，通过qPCR、放射性蛋白质标记和定量实时细胞死亡分析确定，表达VP5的病毒子代大部分能有效离开感染细胞。相比之下，缺乏VP5的病毒子代的释放明显减少，且与细胞死亡相关。综上所述，本报告中的数据表明，IBDV使用了一种先前未描述的依赖VP5的非裂解性出芽机制，显著提高了病毒的传播速度。超微结构分析显示，新组装的IBDV病毒粒子与一个囊泡网络相关联，这显然有助于它们从病毒组装工厂运输到细胞外环境，并且这种关联需要VP5多肽的表达。
