Huang Qinfeng, Shao Junjie, Lan Shuiyun, Zhou Yanqin, Xing Junji, Dong Changjiang, Liang Yuying, Ly Hinh
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Twin Cities, Minnesota, USA Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Twin Cities, Minnesota, USA.
J Virol. 2015 Jul;89(13):6595-607. doi: 10.1128/JVI.00009-15. Epub 2015 Apr 15.
Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFN-deficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication.
Arenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.
沙粒病毒可导致人类严重出血热疾病，针对这些疾病的预防和治疗措施有限。先前对沙粒病毒核蛋白（NP）的结构和功能分析揭示了一个保守的DEDDH核酸外切酶（核糖核酸酶）结构域，该结构域对I型干扰素（IFN）抑制很重要，但NP核糖核酸酶在病毒复制和宿主免疫抑制中的生物学作用尚未得到充分表征。用原型沙粒病毒皮钦德病毒（PICV）感染豚鼠可作为沙粒病毒出血热的替代小动物模型。在本报告中，我们表明PICV NP的五个核糖核酸酶催化残基中的每一个发生突变都会降低IFN抑制活性，并略微降低病毒RNA复制活性。具有核糖核酸酶催化突变的重组PICV可诱导高水平的IFN，并且在具有IFN活性的A549细胞中几乎无法生长，这与野生型（WT）病毒形成鲜明对比，而在缺乏IFN的Vero细胞中，WT和突变病毒都可以相对高水平地复制。感染豚鼠后，核糖核酸酶突变病毒会刺激强烈的IFN反应，无法有效复制，并可能成为WT回复株。核糖核酸酶突变体在体外连续传代也可产生WT回复株。因此，NP核糖核酸酶功能对于允许建立有效的早期病毒感染的先天免疫抑制至关重要，并且它可能部分参与病毒RNA复制过程。
拉沙病毒、卢乔病毒和马丘波病毒等沙粒病毒可导致人类严重且致命的出血热疾病，针对这些疾病的预防和治疗选择有限。广谱抗病毒药物的开发依赖于对病毒感染中保守的沙粒病毒蛋白有更好的机制理解。所有沙粒病毒的核蛋白（NP）都携带一个独特的核酸外切酶（核糖核酸酶）结构域，该结构域已被证明对I型干扰素的抑制至关重要。然而，NP核糖核酸酶在沙粒病毒复制和宿主免疫抑制中的功能作用尚未得到系统表征。我们使用原型沙粒病毒皮钦德病毒（PICV），在细胞培养和豚鼠模型中表征了重组核糖核酸酶缺陷突变体的病毒生长和先天免疫抑制。我们的研究表明，NP核糖核酸酶在抑制宿主先天免疫中起重要作用，可能也参与病毒RNA复制，并且它可以作为开发针对沙粒病毒病原体的抗病毒药物的新靶点。
