Bossart Katharine N, Crameri Gary, Dimitrov Antony S, Mungall Bruce A, Feng Yan-Ru, Patch Jared R, Choudhary Anil, Wang Lin-Fa, Eaton Bryan T, Broder Christopher C
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
J Virol. 2005 Jun;79(11):6690-702. doi: 10.1128/JVI.79.11.6690-6702.2005.
Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae, which are distinguished by their ability to cause fatal disease in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) glycoproteins. Previously, we reported on HeV- and NiV-mediated fusion activities and detailed their host-cell tropism characteristics. These studies also suggested that a common cell surface receptor, which could be destroyed by protease, was utilized by both viruses. To further characterize the G glycoprotein and its unknown receptor, soluble forms of HeV G (sG) were constructed by replacing its cytoplasmic tail and transmembrane domains with an immunoglobulin kappa leader sequence coupled to either an S-peptide tag (sG(S-tag)) or myc-epitope tag (sG(myc-tag)) to facilitate purification and detection. Expression of sG was verified in cell lysates and culture supernatants by specific affinity precipitation. Analysis of sG by size exclusion chromatography and sucrose gradient centrifugation demonstrated tetrameric, dimeric, and monomeric species, with the majority of the sG released as a disulfide-linked dimer. Immunofluorescence staining revealed that sG specifically bound to HeV and NiV infection-permissive cells but not to a nonpermissive HeLa cell line clone, suggesting that it binds to virus receptor on host cells. Preincubation of host cells with sG resulted in dose-dependent inhibition of both HeV and NiV cell fusion as well as infection by live virus. Taken together, these data indicate that sG retains important native structural features, and we further demonstrate that administration of sG to rabbits can elicit a potent cross-reactive neutralizing antibody response against infectious HeV and NiV. This HeV sG glycoprotein will be exceedingly useful for structural studies, receptor identification strategies, and vaccine development goals for these important emerging viral agents.
亨德拉病毒（HeV）和尼帕病毒（NiV）是密切相关的新兴病毒，属于副粘病毒亚科的亨尼帕病毒属，其特点是能够在动物和人类宿主中引发致命疾病。这些病毒通过其附着（G）糖蛋白和融合（F）糖蛋白介导的不依赖pH的膜融合事件感染细胞。此前，我们报道了HeV和NiV介导的融合活性，并详细阐述了它们的宿主细胞嗜性特征。这些研究还表明，两种病毒都利用了一种可被蛋白酶破坏的共同细胞表面受体。为了进一步表征G糖蛋白及其未知受体，通过用与S肽标签（sG(S-tag)）或myc表位标签（sG(myc-tag)）偶联的免疫球蛋白κ前导序列替换其胞质尾和跨膜结构域，构建了可溶性形式的HeV G（sG），以促进纯化和检测。通过特异性亲和沉淀在细胞裂解物和培养上清液中验证了sG的表达。通过尺寸排阻色谱和蔗糖梯度离心对sG进行分析，结果显示有四聚体、二聚体和单体形式，其中大部分sG以二硫键连接的二聚体形式释放。免疫荧光染色显示，sG特异性结合HeV和NiV感染允许细胞，但不结合非允许的HeLa细胞系克隆，这表明它与宿主细胞上的病毒受体结合。用sG对宿主细胞进行预孵育会导致HeV和NiV细胞融合以及活病毒感染受到剂量依赖性抑制。综上所述，这些数据表明sG保留了重要的天然结构特征，并且我们进一步证明，给兔子注射sG可以引发针对传染性HeV和NiV的强效交叉反应性中和抗体反应。这种HeV sG糖蛋白对于这些重要新兴病毒病原体的结构研究、受体鉴定策略和疫苗开发目标将非常有用。
