Molecular Requirements for Self-Interaction of the Respiratory Syncytial Virus Matrix Protein in Living Mammalian Cells.

Marta Trevisan,Reena Ghildyal,Annalisa Radeghieri,Gualtiero Alvisi,Veronica Di Antonio,Giorgio Palù

doi:10.3390/v10030109

Abstract

Respiratory syncytial virus (RSV) is an important human pathogen, which infects respiratory tract epithelial cells causing bronchiolitis and pneumonia in children and the elderly. Recent studies have linked RSV matrix (M) ability to self-interaction and viral budding. However, RSV M has been crystalized both as a monomer and a dimer, and no formal proof exists to date that it forms dimers in cells. Here, by using a combination of confocal laser scanning microscopy and bioluminescent resonant energy transfer applied to differently tagged deletion mutants of RSV M, we show that the protein can self-interact in living mammalian cells and that both the N and C-terminus of the protein are strictly required for the process, consistent with the reported dimeric crystal structure.

Highlights

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants and young children [1,2,3], responsible for one-third of deaths resulting from acute lower respiratory infection in the first year of life [4,5,6]
RSV M deletion mutants as expressed in Mammalian cells when C-terminally fused to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)
Our results indicate that the Renilla luciferase (RLuc)-YFP fusion generated a bioluminescent resonant energy transfer (BRET) ratio of 0.34 ± 0.02 (Figure 4A), while the RLuc and YFP

Summary

Introduction

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants and young children [1,2,3], responsible for one-third of deaths resulting from acute lower respiratory infection in the first year of life [4,5,6]. There are no vaccines or antiviral drugs that effectively target RSV despite decades of research [8]. Deeper understanding of the molecular mechanisms that underlie RSV assembly could pave the way to the identification of new vaccine/antiviral targets. RSV is an enveloped virus with a non-segmented negative sense RNA genome and belongs to the Orthopneumovirus genus of the Pneumoviridae family [9]. The RSV genome is tightly encapsidated within the nucleocapsid, which is composed of nucleocapsid protein N, the RNA polymerase L and its cofactor phosphoprotein P, as well as the M2-1 protein. External to the nucleocapsid is a layer of matrix (M) protein which acts as a bridge between the nucleocapsid and the lipid bilayer envelope

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