Acciani Marissa, Alston Jacob T, Zhao Guohui, Reynolds Hayley, Ali Afroze M, Xu Brian, Brindley Melinda A
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA.
Department of Infectious Diseases, Department of Population Health, Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA
J Virol. 2017 Aug 24;91(18). doi: 10.1128/JVI.00574-17. Print 2017 Sep 15.
Lassa virus (LASV) is an enveloped RNA virus endemic to West Africa and responsible for severe cases of hemorrhagic fever. Virus entry is mediated by the glycoprotein complex consisting of a stable-signal peptide, a receptor-binding subunit, GP1, and a viral-host membrane fusion subunit, GP2. Several cellular receptors can interact with the GP1 subunit and mediate viral entry, including alpha-dystroglycan (αDG) and lysosome-associated membrane protein 1 (LAMP1). In order to define the regions within GP1 that interact with the cellular receptors, we implemented insertional mutagenesis, carbohydrate shielding, and alanine scanning mutagenesis. Eighty GP constructs were engineered and evaluated for GP1-GP2 processing, surface expression, and the ability to mediate cell-to-cell fusion after low-pH exposure. To examine virus-to-cell entry, 49 constructs were incorporated onto vesicular stomatitis virus (VSV) pseudoparticles and transduction efficiencies were monitored in HAP1 and HAP1-ΔDAG1 cells that differentially produce the αDG cell surface receptor. Seven constructs retained efficient transduction in HAP1-ΔDAG1 cells yet poorly transduced HAP1 cells, suggesting that they are involved in αDG utilization. Residues H141, N146, F147, and Y150 cluster at the predicted central core of the trimeric interface and are important for GP-αDG interaction. Additionally, H92A-H93A, 150HA, 172HA, and 230HA displayed reduced transduction in both HAP1 and HAP1-ΔDAG1 cells, despite efficient cell-to-cell fusion activity. These mutations may interfere with interactions with the endosomal receptor LAMP1 or interfere at another stage in entry that is common to both cell lines. Insight gained from these data can aid in the development of more-effective entry inhibitors by blocking receptor interactions. Countries in which Lassa virus is endemic, such as Nigeria, Sierra Leone, Guinea, and Liberia, usually experience a seasonal outbreak of the virus from December to March. Currently, there is neither a preventative vaccine nor a therapeutic available to effectively treat severe Lassa fever. One way to thwart virus infection is to inhibit interaction with cellular receptors. It is known that the GP1 subunit of the Lassa glycoprotein complex plays a critical role in receptor recognition. Our results highlight a region within the Lassa virus GP1 protein that interacts with the cellular receptor alpha-dystroglycan. This information may be used for future development of new Lassa virus antivirals.
拉沙病毒（LASV）是一种包膜RNA病毒，在西非流行，可导致严重的出血热病例。病毒进入细胞是由糖蛋白复合体介导的，该复合体由一个稳定信号肽、一个受体结合亚基GP1和一个病毒-宿主膜融合亚基GP2组成。几种细胞受体可与GP1亚基相互作用并介导病毒进入，包括α- dystroglycan（αDG）和溶酶体相关膜蛋白1（LAMP1）。为了确定GP1中与细胞受体相互作用的区域，我们进行了插入诱变、碳水化合物屏蔽和丙氨酸扫描诱变。构建了80个GP构建体，并对其进行了GP1 - GP2加工、表面表达以及低pH暴露后介导细胞间融合能力的评估。为了检测病毒到细胞的进入过程，将49个构建体整合到水泡性口炎病毒（VSV）假病毒颗粒上，并在差异表达αDG细胞表面受体的HAP1和HAP1 -ΔDAG1细胞中监测转导效率。7个构建体在HAP1 -ΔDAG1细胞中保留了高效转导能力，但在HAP1细胞中转导效率很低，这表明它们参与了αDG的利用。残基H141、N146、F147和Y150聚集在三聚体界面的预测中心核心处，对GP -αDG相互作用很重要。此外，H92A - H93A、I50HA、172HA和230HA在HAP1和HAP1 -ΔDAG1细胞中的转导效率均降低，尽管它们具有高效的细胞间融合活性。这些突变可能会干扰与内体受体LAMP1的相互作用，或在两个细胞系共有的进入过程的另一个阶段产生干扰。从这些数据中获得的见解有助于通过阻断受体相互作用来开发更有效的进入抑制剂。拉沙病毒流行的国家，如尼日利亚、塞拉利昂、几内亚和利比里亚，通常在12月至3月期间经历该病毒的季节性爆发。目前，既没有预防性疫苗，也没有可有效治疗严重拉沙热的疗法。阻止病毒感染的一种方法是抑制与细胞受体的相互作用。已知拉沙糖蛋白复合体的GP1亚基在受体识别中起关键作用。我们的结果突出了拉沙病毒GP1蛋白中与细胞受体α- dystroglycan相互作用的一个区域。这些信息可用于未来开发新的拉沙病毒抗病毒药物。
