Mutation of the TYTLE Motif in the Cytoplasmic Tail of the Sendai Virus Fusion Protein Deeply Affects Viral Assembly and Particle Production

Manel Essaidi-Laziosi,Anastasia Shevtsova,Denis Gerlier,Laurent Roux

doi:10.1371/journal.pone.0078074

Abstract

Enveloped viruses contain glycoproteins protruding from the viral membrane. These proteins play a crucial role in the extra-cellular steps of the virus life cycle, namely attachment to and entry into cells. Their role during the intracellular late phase of virus multiplication has been less appreciated, overlooked by the documented central organizer role of the matrix M protein. Sendai virus, a member of the Paramyxoviridae family, expresses two trans-membrane proteins on its surface, HN and F. In previous work, we have shown that suppression of F in the context of an infection, results in about 70% reduction of virus particle production, a reduction similar to that observed upon suppression of the matrix M protein. Moreover, a TYTLE motif present in F cytoplasmic tail has been proposed essential for virus particle production. In the present work, using original alternate conditional siRNA suppression systems, we generated a double F gene recombinant Sendai virus expressing wt-F and a nonviable mutated TYTLE/5A F protein (F5A). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F5A i) was not expressed at the cell surface but restricted to the endoplasmic reticulum, ii) was still capable of interaction with M and iii) had profound effect on M and HN cellular distribution. On the basis of these data, we propose a model for SeV particle formation based on an M/F complex that would serve as nucleation site for virus particle assembly at the cell surface.

Highlights

Enveloped viruses contain trans-membrane glycoproteins that protrude from the particle envelope
By comparison with rSeV-HA-F/Fgfpt, rSeV-HA-F5A/Fgfpt virions were devoid of the HA-F1 upper band (Figure 3A, HA-F1) and upon infection of MDCK cells, only the lower wt F band sensitive to a-gfpt siRNA silencing could be detected with only trace amount of a-HA reactive HA-F5A band
When the TYTLE motif present in the cytoplasmic domain of SeV-F was mutated to 5 alanines (5A), no virus could be rescued

Summary

Introduction

Enveloped viruses contain trans-membrane glycoproteins that protrude from the particle envelope. The glycoproteins shortly extend from the inner side of the envelope to contact the matrix protein and/or the viral core These so-called cytoplasmic tails (ct) vary in size, ranging from a few amino acid residues to several tens. They allow attachment of the particle by binding to specific cellular receptors and they orchestrate the fusion of the viral envelope with the cellular membrane to deliver the viral genome inside the cell. To these essential roles, the involvement of the glycoproteins in virus particle formation and production has been recognized, namely their participation in the formation of the assembly complex at the membrane, as well as in the induction of the membrane curvature leading to the formation of the viral bud (pull effect, for a review on the subject see Welsch et al [1]). A critical role for the hemagglutinin-neuraminidase (HN) protein of the Parainfluenza virus type 5 (PIV5) was reported, emphasizing the involvement of its cytoplasmic tail domain at the expense of the M and, interestingly, of the other glycoprotein F [8,9,10]

Methods

Results

Conclusion