Respiratory syncytial virus morphogenesis in the human nasal epithelium
The nasopharyngeal cavity is a primary route of respiratory syncytial virus (RSV) infection. There is currently relatively little information about RSV morphogenesis in the nasal epithelium, which is a prerequisite for understanding RSV transmission in the upper respiratory tract. We therefore examined the cilia and RSV particle distribution on RSV-infected nasal epithelial cells using an air-liquid interface (ALI) culture over the course of 5 days. This ALI culture system is a stratified epithelium with apical ciliated and secretory cells typical of proximal airway. Although RSV infection was confined to ciliated cells within the stratified epithelium, virus morphogenesis occurred at cell surface locations that were distinct from cilia. Virus particles formed on the apical cell surface exhibited a filamentous morphology that was clearly distinct from the larger cilia. However, the F and G virus glycoproteins were also trafficked into the cilia, which correlated with impaired cilia function and cilia loss.
In permissive cell lines RSV particles assemble as filamentous projections called virus filaments (VFs), and inhibiting the formation of VFs restricts RSV transmission. The VFs contain high levels of actin, and their involvement in an actin-based motility system for mediating virus transmission has been proposed. Understanding the molecular mechanism of RSV particle assembly should therefore improve our understanding of the RSV transmission process. Consequently, the motivation for this current study was to design an experimental procedure to test the hypothesis that VFs have a clinical relevance. Using stem cell technology we were able to replicate the human nasal epithelium in a tissue culture dish, and use this cell system to examine the process of RSV morphogenesis in a more ‘clinical’ context. We rationalized that the most direct way to examine virus morphogenesis was to image the virus particles on infected cells. Only the ciliated epithelial cells were infected by RSV, and we were able to demonstrate that VFs formed on the cell surface, and that their presence coincided with cilia dysfunction. Although the virus glycoproteins were trafficked into the cilia, the virus glycoproteins and polymerase-associated proteins accumulated at non-cilia locations. This indicated, much to our surprise, that RSV morphogenesis on ciliated epithelial cells did not occur on the cilia. Using this cell model of infection we can speculate that in the clinical scenario RSV particle assembly induces cilia dysfunction in the nasal cavity, and that this may be a factor in its colonization of the upper respiratory tract.
Loss of (A) cilia beat frequency (%of mock-infected cells) and (B) cilia on mock-infected and virus-infected cells at 5 days post-infection (highlighted by white arrows). Cells were co-stained with anti-N (RSV N protein) and anti-ß4 tubulin (cilia). (C) Scanning electron micrograph showing cilia on mock-infected cells and virus particles (vp) on RSV-infected cells at 5 days (x25,000 magnification).
Introducing the authors
1. Dr. Muhammad Raihan Jumat was a research scientist in the School of Biological Sciences, Nanyang Technological University, Singapore.
2. Dr. Yan Yan is a research scientist with the Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
3. Group photo
(Back row, left to right) Dr. De Yun Wang is a clinician scientist at the Yong Loo Lin School of Medicine, National University Health System, National University of Singapore; Singapore. Dr. Richard Sugrue is a Virologist at the School of Biological Sciences, Nanyang Technological University, Singapore and Dr. Chunwei Li is a research scientist with the Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
(Front row, left to right)
Ms. Tra Nguyen Huong is a student in the School of Biological Sciences, Nanyang Technological University, Dr. Laxmi Iyer Ravi was a student in the School of Biological Sciences, Nanyang Technological University; Singapore. Dr. Boon Huan Tan is a virologist and program director at the Detection and Diagnostics Laboratory, DSO National Laboratories, Singapore and Ms. Puisan Wong is a technical specialist in the Detection and Diagnostics Laboratory, DSO National Laboratories, Singapore.
About the research
Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia
Virology, Volume 484, October 2015, Pages 395–411
Muhammad Raihan Jumat, Yan Yan, Laxmi Iyer Ravi, Puisan Wong, Tra Nguyen Huong, Chunwei Li, Boon Huan Tan, De Yun Wang, Richard J. Sugrue