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

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.

Discrete spatio-temporal regulation of tyrosine phosphorylation directs influenza A virus M1 protein towards its function in virion assembly

“…Here, prevention of M1 Y132 phosphorylation led to protein aggregation at the plasma membrane in late stages of infection. This accumulation in patches at the cell periphery has been reported previously for mutations of aa R76-78 [18,19]. This triplet belongs to the patch of residues with positive charge, which was shown to be directly involved in M1 association with membranes and in M1-M1 interactions [20,40,41].…”

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

“…Furthermore, it is known that the association between M1 and lipids is driven mainly by electrostatic forces and that the protein interacts with negatively charged lipids, in particular with phosphatidylserine (PS) (6,7,10). More specifically, targeting of M1 to the PM of infected cells might rely on interactions with other viral proteins (e.g., M2 or HA) and/or lipid domains (5,(11)(12)(13)(14)(15). In fact, a long-standing hypothesis is that viral budding occurs at specific regions of the PM, namely, lipid rafts, i.e., lipid domains enriched in sphingolipids and cholesterol (5,(16)(17)(18)(19)(20)(21).…”

The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model

“…In addition, positively charged residues of M1-A (R76/77/78) are required for M1 membrane targeting and attachment as well as incorporation into virions3334. However, the basic surface may not suffice for membrane interaction in vivo 35.…”