Kiss Gabriella, Holl Jens M, Williams Grant M, Alonas Eric, Vanover Daryll, Lifland Aaron W, Gudheti Manasa, Guerrero-Ferreira Ricardo C, Nair Vinod, Yi Hong, Graham Barney S, Santangelo Philip J, Wright Elizabeth R
Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
J Virol. 2014 Jul;88(13):7602-17. doi: 10.1128/JVI.00256-14. Epub 2014 Apr 23.
Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization.
hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles. Depending on the virus morphology examined, the amount of M ranged from 24% to 86%. We complemented the cryo-imaging studies with fluorescence microscopy, dSTORM, and a proximity ligation assay to provide additional evidence that M2-1 is incorporated into viral particles and is positioned between M and RNP. The results highlight the impact of M and M2-1 on the regulation of hRSV organization.
呼吸道合胞病毒（RSV）是副粘病毒科的成员，属于非节段、负链、单链RNA基因组病毒，是婴儿、幼儿、老年人或免疫功能低下者下呼吸道感染的主要原因。关于调节人呼吸道合胞病毒（hRSV）组装和出芽的过程，仍有许多悬而未决的问题。在这里，我们使用冷冻电子断层扫描技术，在同一病毒制剂中鉴定出了结构呈球形、丝状和不对称的病毒颗粒。这三种颗粒形态的表面糖蛋白、基质蛋白（M）、M2-1和核糖核蛋白（RNP）保持相似的组织方式。对RNP细丝进行了三维（3D）追踪，并计算了它们的总长度。测量结果显示每个颗粒包含多个全长基因组拷贝。只要存在M层，RNP就与膜相关联。在不同形态中，M的覆盖量范围为24%至86%。使用荧光显微镜（fLM）、直接随机光学重建显微镜（dSTORM）和邻近连接分析（PLA），我们提供了证据表明，在分离的病毒颗粒中，M2-1位于RNP和M之间。此外，当使用泽尔尼克相衬（ZPC）冷冻电子断层扫描对hRSV病毒进行成像时，解析出了M2-1密度的规则间距。我们的研究对hRSV病毒体结构进行了更完整的表征，并证实M和M2-1调节病毒组织。
hRSV是婴儿、幼儿以及老年人或免疫功能低下个体下呼吸道感染的主要原因。我们使用冷冻电子断层扫描和泽尔尼克相衬冷冻电子断层扫描对纯化的hRSV群体进行三维可视化。我们观察到三种不同的形态，球形、丝状和不对称，它们保持了可比的组织轮廓。根据所检查的病毒形态，M的量范围为24%至86%。我们用荧光显微镜、dSTORM和邻近连接分析对冷冻成像研究进行补充，以提供额外证据表明M2-1被整合到病毒颗粒中，并位于M和RNP之间。结果突出了M和M2-1对hRSV组织调节的影响。
