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.
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.
Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS & Université of Montpellier, Montpellier, France.
Adeline
A
Kerviel
Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS & Université of Montpellier, Montpellier, France.
Shantoshini
S
Dash
Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS & Université of Montpellier, Montpellier, France.
Olivier
O
Moncorgé
CIRI, INSERM U 1111, France & ENS de Lyon, Lyon, France.
Baptiste
B
Panthu
Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS & Université of Montpellier, Montpellier, France.
Jan
J
Prchal
CIRI, INSERM U 1111, France & ENS de Lyon, Lyon, France.
Didier
D
Décimo
CIRI, INSERM U 1111, France & ENS de Lyon, Lyon, France.
Théophile
T
Ohlmann
Université de Lyon, Université Lyon 1, Faculté de Médecine Lyon Est, Laboratoire de Virologie et Pathologie Humaine, EA 4610, Lyon, France.
Bruno
B
Lina
Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS & Université of Montpellier, Montpellier, France.
Cyril
C
Favard
Aix-Marseille Université & CNRS, AFMB UMR 7257, 163 Avenue de Luminy, 13288 Marseille cedex 09, France.
Etienne
E
Decroly
Université de Lyon, Université Lyon 1, Faculté de Médecine Lyon Est, Laboratoire de Virologie et Pathologie Humaine, EA 4610, Lyon, France.
Michèle
M
Ottmann
INSERM U966, Université François Rabelais & CHRU de Tours, Tours, France.
Philippe
P
Roingeard
Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS & Université of Montpellier, Montpellier, France.
Delphine
D
Muriaux
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D057809:HEK293 Cells; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000378:metabolism*; D007251:Influenza, Human / Q000821:virology; D061985:Madin Darby Canine Kidney Cells; D009154:Mutation / Q000235:genetics; D019913:Nuclear Localization Signals / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology
D000596:Amino Acids; C488936:M1 protein, Influenza A virus; D019913:Nuclear Localization Signals; D014763:Viral Matrix Proteins; D001120:Arginine