Monaghan Paul, Green Diane, Pallister Jackie, Klein Reuben, White John, Williams Catherine, McMillan Paul, Tilley Leann, Lampe Marko, Hawes Pippa, Wang Lin-Fa
CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia.
Department of Biochemistry and Molecular Biology, Melbourne, Australia.
Virol J. 2014 Nov 27;11:200. doi: 10.1186/s12985-014-0200-5.
Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.
A range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy.
Significant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions.
These findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.
亨德拉病毒（HeV）是一种属于副粘病毒科的多形性病毒。我们的长期目标是了解HeV病毒粒子的组装过程。作为第一步，我们试图确定用于研究此过程的最合适细胞培养系统，然后使用该模型来定义病毒的形态，并通过对感染细胞中关键病毒编码蛋白进行成像来确定组装位点。
用HeV感染一系列原代细胞和永生化细胞系，在感染后的不同时间点固定，标记HeV蛋白，并通过共聚焦显微镜、超分辨率显微镜和透射电子显微镜成像。
根据感染的细胞类型，病毒蛋白分布存在显著差异。在感染后8小时，在内质网中检测到HeV G蛋白，在所有测试细胞的细胞核中主要观察到M蛋白。在感染后18小时，感染HeV的Vero细胞在整个细胞中显示出M和G蛋白，并且在透射电子显微镜（TEM）切片中，在多形性病毒样结构中也有显示。在感染HeV的MDBK、A549和HeLa细胞中，HeV M蛋白主要见于细胞核，G蛋白位于细胞膜。在感染HeV的原代牛和猪主动脉内皮细胞以及两种蝙蝠来源的细胞系中，在任何测试时间点（感染后8、12、18、24、48小时），HeV M蛋白在细胞核中的水平都没有那么高，但主要在细胞表面观察到，呈点状模式，与G蛋白共定位。通过TEM和超分辨率（SR）显微镜证实这些HeV M和G阳性结构为圆形HeV病毒粒子。SR成像首次展示了副粘病毒蛋白的亚病毒粒子成像以及HeV G、M和N蛋白在病毒粒子内的各自定位。
这些发现为HeV的结构提供了新的见解，并表明对于HeV成像研究，组织培养细胞的选择可能会影响实验结果。结果还表明，通过TEM，HeV应被视为主要为圆形的病毒，平均直径约为280nm，通过SR成像则为310nm。
