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 1 , Anastasia Shevtsova 1 , Denis Gerlier 2 , Laurent Roux 1 * 1 Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland, 2 Centre International de Recherche en Infectiologie, Universite´ Lyon 1, ENS de Lyon, Lyon, France 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 (F 5A ). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F 5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F 5A 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. Citation: Essaidi-Laziosi M, Shevtsova A, Gerlier D, Roux L (2013) Mutation of the TYTLE Motif in the Cytoplasmic Tail of the Sendai Virus Fusion Protein Deeply Affects Viral Assembly and Particle Production. PLoS ONE 8(12): e78074. doi:10.1371/journal.pone.0078074 Editor: Paul Digard, University of Edinburgh, United Kingdom Received July 3, 2013; Accepted September 17, 2013; Published December 10, 2013 Copyright: ß 2013 Essaidi-Laziosi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was carried out with the financial support of the Swiss National Foundation for Scientific Research (to LR) and of the Agence Nationale de la Recherche, France, specific program ‘‘Physico-Chimie du Vivant’’, ANR-08-PCVI-0020-01 (to DG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: laurent.roux@unige.ch Introduction Enveloped viruses contain trans-membrane glycoproteins that protrude from the particle envelope. The glycoproteins also 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. Upon infection, these glycoproteins have a dual role. 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 also 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]). In view of the well-established role of the matrix protein M described as the central organizer of the assembly complex, the implication of the glycoproteins in these late steps has gradually grown in impor- tance, although there are variations depending on the virus. More specifically, and as examples, the HIV Env appears dispensable as Gag alone buds very efficiently [2] [3] [4] [5]. This contrasts with the 30 fold increased budding of rabies virus like particles (VLP) promoted by its glycoprotein G [6]. Similarly, the influenza virus hemagglutinin (HA) and neuraminidase (NA) have been recog- nized more compulsory for VLP production than the matrix protein M1 [7]. A critical role for the hemagglutinin-neuramin- idase (HN) protein of the Parainfluenza virus type 5 (PIV5) was equally reported, emphasizing the involvement of its cytoplasmic tail domain at the expense of the M and, interestingly, of the other glycoprotein F [8–10]. Sendai virus (SeV), as influenza virus or PIV5, contains two glycoproteins protruding on its envelope, HN and F, with, respectively, receptor binding/cleavage and viral envelope/cellu- lar membrane fusion activities. To evaluate the importance of these two surface glycoproteins in the process of particle production, recombinant Sendai viruses (rSeV) were produced with HN and F harboring gradual truncations of, as well as site directed mutations in their cytoplasmic tail [11]. For HN (type II glycoprotein, ct = 35 aa at the N-terminus), a motif 10 SYWST 14 , described previously as required for HN incorporation into virions [12], was confirmed in this function. However, production of normal level of virus particles with undetectable amount of HN was found possible, making HN dispensable for virus particle production. For F (type I glycoprotein, ct = 42 aa at the PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e78074