IHEM, Universidad Nacional de Cuyo, CONICET, Facultad de Ciencias Médicas, Mendoza, Argentina.
Facultad de Ciencias Veterinarias y Ambientales, Universidad Juan Agustín Maza, Mendoza, Argentina.
J Virol. 2018 May 14;92(11). doi: 10.1128/JVI.01964-17. Print 2018 Jun 1.
Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses. Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.
双贝类病毒是双链 RNA (dsRNA) 病毒组的非常规成员，其特征是缺乏转录活性的内核。相反，双贝类病毒颗粒将其基因组组织在核糖核蛋白复合物 (RNP) 中，该复合物由 dsRNA 片段、dsRNA 结合 VP3 蛋白和病毒编码的 RNA 依赖性 RNA 聚合酶 (RdRp) 组成。这些和其他结构特征表明，双贝类病毒可能遵循与家族成员完全不同的复制程序，支持双贝类病毒是单链正 RNA(+ssRNA) 和 dsRNA 病毒之间进化联系的假说。在这里，我们证明了传染性法氏囊病病毒 (IBDV)，一种 家族的典型成员，通过病毒蛋白 VP3 与这些隔室胞质小叶上的磷脂相互作用，劫持感染细胞的内体膜进行复制。通过使用诱变方法，我们证明 VP3 结构域 PATCH 2 (P2) 介导 VP3 与内体膜的结合。为了确定 VP3 P2 在病毒复制周期中的作用，我们使用稳定过表达 VP3 P2 的禽细胞进行 IBDV 感染。重要的是，稳定过表达 VP3 P2 的细胞中，细胞内外病毒产量以及细胞内 VP2 病毒衣壳蛋白水平均显著降低。总之，我们的结果表明，VP3 与内体的结合在 IBDV 复制周期中具有重要作用。本报告为 dsRNA 病毒的复制提供了内体等膜性隔室是必需的直接实验证据。该结果还支持了先前提出的双贝类病毒作为+ssRNA 和 dsRNA 病毒之间进化联系的假说。传染性法氏囊病 (IBD；也称为 Gumboro 病) 是一种影响雏鸡的急性、高度传染性的免疫抑制性疾病，在全球范围内传播。IBD 的病原体是传染性法氏囊病病毒 (IBDV)。该病毒破坏中枢免疫器官 (法氏囊)，导致免疫抑制和鸡对疫苗的反应降低，从而增加其对其他病原体的易感性。IBDV 是 家族的成员，该家族包括 dsRNA 病毒的非常规成员，其复制策略研究甚少。在本报告中，我们表明 IBDV 通过 RNP 复合物成分 VP3 与内体膜胞质小叶中的磷脂结合，劫持感染细胞的内体，为建立病毒复制复合物。我们表明这种相互作用由 VP3 PATCH 2 结构域介导，并证明其在病毒感染背景下的相关作用。
