Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus.

Adeline Kerviel,Didier Décimo,Cyril Favard,Théophile Ohlmann,Delphine Muriaux,Baptiste Panthu,Philippe Roingeard,Olivier Moncorgé,Etienne Decroly,Shantoshini Dash,Jan Prchal,Bruno Lina,Michèle Ottmann

doi:10.1371/journal.pone.0165421

Abstract

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity.

Highlights

The influenza A(H1N1)pdm09 strain spread in 2009 and caused the first influenza pandemic of the 21st century
Using our minimal experimental system with the addition of segment 7 (M) for optimal M1 membrane detection (i.e., M1+M2+Non Structural protein 1 (NS1)/NEP+M), we investigated the effect of mutations in the M1 arginine triplet (M1 R76/77/78A) or in the Nuclear Localization Signal (NLS) (K101/102A) on M1 attachment to cell membranes by quantification following western blot analysis of the different fractions obtained by subcellular fractionation, as described in Materials and Methods
Our results suggest that M2 and NS1/NEP are the minimum viral determinants for M1 membrane localization in transfected cells

Summary

Introduction

The influenza A(H1N1)pdm strain spread in 2009 and caused the first influenza pandemic of the 21st century. The influenza A virus is composed of eight viral RNA segments (PB2, PB1, PA, HA, NP, NA, M and NS) that encode ten major proteins. And budding of influenza virions is a multi-step process that occurs at the cell plasma membrane of infected cells [1]. The virus "core" includes the eight viral ribonucleoprotein (vRNP) complexes each of which is composed of one viral RNA segment that encodes one or more viral proteins coated by nucleoproteins (NP). This “core” is complexed with a polymerase complex made of three subunits (PB1, PB2, and PA). M1 has a central role in the assembly and release of viral particles, as indicated by the finding that both processes are abrogated in its absence [5]

Methods

Results

Conclusion