Identification of RSV Fusion Protein Interaction Domains on the Virus Receptor, Nucleolin.

Peter Mastrangelo,Amy H Jeon,Karen K Siu,Cameron A Ackerley,Jeffrey E Lee,Richard G Hegele,Stephanie Tan,Allysia A Chin

doi:10.3390/v13020261

Peter Mastrangelo, Amy H Jeon + Show 6 more

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https://doi.org/10.3390/v13020261

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Journal: Viruses	Publication Date: Feb 8, 2021
Citations: 22	License type: CC BY 4.0

Affiliation: University of Toronto, Hospital for Sick Children

Abstract

Nucleolin is an essential cellular receptor to human respiratory syncytial virus (RSV). Pharmacological targeting of the nucleolin RNA binding domain RBD1,2 can inhibit RSV infections in vitro and in vivo; however, the site(s) on RBD1,2 which interact with RSV are not known. We undertook a series of experiments designed to: document RSV-nucleolin co-localization on the surface of polarized MDCK cells using immunogold electron microscopy, to identify domains on nucleolin that physically interact with RSV using biochemical methods and determine their biological effects on RSV infection in vitro, and to carry out structural analysis toward informing future RSV drug development. Results of immunogold transmission and scanning electron microscopy showed RSV-nucleolin co-localization on the cell surface, as would be expected for a viral receptor. RSV, through its fusion protein (RSV-F), physically interacts with RBD1,2 and these interactions can be competitively inhibited by treatment with Palivizumab or recombinant RBD1,2. Treatment with synthetic peptides derived from two 12-mer domains of RBD1,2 inhibited RSV infection in vitro, with structural analysis suggesting these domains are potentially feasible for targeting in drug development. In conclusion, the identification and characterization of domains of nucleolin that interact with RSV provide the essential groundwork toward informing design of novel nucleolin-targeting compounds in RSV drug development.

Highlights

According to the World Health Organization (WHO), respiratory syncytial virus (RSV), or human orthopneumovirus, is a leading cause of acute lower respiratory infection and hospitalization in infants and young children [1]
We showed that interaction of nucleolin with RSV, in particular the RSV fusion protein (RSVF), was required for infection, and when nucleolin was silenced via siRNA, RSV infection was reduced
RSV-infected MDCK cells were prepared for Transmission Electron Microscopy (TEM) and double labeled using immunogold for RSV and nucleolin to visualize co-localization at the cell surface, as expected for a viral receptor (Figure 1)

Summary

Introduction

According to the World Health Organization (WHO), respiratory syncytial virus (RSV), or human orthopneumovirus, is a leading cause of acute lower respiratory infection and hospitalization in infants and young children [1]. After almost 60 years of research, no vaccine is currently available [2] In this time of the COVID-19 pandemic, the possibility looms that we may, in the coming flu season, have to deal with a resurgence of COVID19, the influenza virus, and RSV simultaneously [3]. We showed that targeting nucleolin with the DNA aptamer, AS1411, originally developed as a cancer therapeutic, could reduce infection of cells in culture, but in two animal models with a corresponding reduction of lung inflammation [5]. It has been shown that targeting a pathway that controls shuttling of nucleolin to the cell surface is a viable strategy to reduce RSV infection [6]

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Conclusion