The Lack of an Inherent Membrane Targeting Signal Is Responsible for the Failure of the Matrix (M1) Protein of Influenza A Virus To Bud into Virus-Like Particles

Abstract

The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.The late phase of the influenza A virus replication cycle is marked by the occurrence of assembly and budding at the plasma membrane of infected cells, which leads to the separation of virion and host cell membranes and ultimately results in the production of infectious virus particles. This critical step is a highly concerted process driven largely by protein-protein, protein-lipid, and protein-nucleic acid interactions (34, 40). It has been established for many years that four viral structural components, namely, the matrix protein (M1), hemagglutinin (HA), neuraminidase (NA), and M2, are actively involved in the assembly and budding process (34,35,40), although the identities of these inter-and intramolecular interactions and regulatory mechanisms for influenza A virus assembly and budding are unclear. It has also been suggested that interactions of M1 with various cytoplasmic tails (CTs) of HA, NA, and M2 are critical to drive the assembly and release of influenza A virions from the surface of infected cells (1,5,10,18,25,29,30,68). To date, these interactions have been largely speculative because direct interactions have been demonstrated only for M1 and M2 (5, 18, 29).Early investigations into the budding machinery of influenza A virus using vaccinia virus-and baculovirus-based expression systems indicated that M1 was the only viral protein absolutely required for the assembly of virus particles (14,15...

Formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase

“…1b); however, the amount of M1 released with the budding VLPs in the supernatant was approximately half that found in virus-infected cells. This is compatible with previous reports that M1 requires interaction with other viral proteins, such as the structural proteins HA/NA (Wang et al, 2010), as well as with vRNPs, which are absent from VLPs. Using TEM, the VLPs were seen to be pleomorphic, with spherical or filamentous forms (Fig.…”

“…Gó mezPuertas et al (2000) suggested that M1 plays the major role in driving virus budding from the cellular membrane, whereas Chen et al (2007) found that M1 was not essential for the process, but that HA and NA were necessary. A recent publication (Wang et al, 2010) confirmed that M1 by itself fails to form VLPs, probably due to a lack of membrane-targeting signal, and therefore requires interaction with other viral proteins to be incorporated into the budding virions. In our study, although small amounts of M1 were released from cells expressing M1 alone, no budding particles were observed by TEM (Fig.…”

The Amphipathic Helix of Influenza A Virus M2 Protein Is Required for Filamentous Bud Formation and Scission of Filamentous and Spherical Particles

“…M2 protein associates mainly with the periphery of lipid rafts and is largely excluded from virions. In contrast, M1 cannot target the plasma membrane in the absence of other viral proteins (10,11). M1 expressed alone in cells cannot form virus-like particles (12) and likely targets the plasma membrane by associating with the cytoplasmic tails of the viral transmembrane proteins such as HA, NA (13,14), and M2 (11,15).…”

The Highly Conserved Arginine Residues at Positions 76 through 78 of Influenza A Virus Matrix Protein M1 Play an Important Role in Viral Replication by Affecting the Intracellular Localization of M1

“…Early studies using various expression systems showed that M1 alone could form virus-like particles (VLPs) (12,21). In contrast, recent work by Chen et al (7) and Wang et al (47) suggests that M1 cannot form VLPs by itself and that other viral proteins are required. Wang et al also showed that M1 does not have an inherent membrane targeting signal and is trafficked to the plasma membrane with the help of M2.…”

“…Wang et al also showed that M1 does not have an inherent membrane targeting signal and is trafficked to the plasma membrane with the help of M2. M1 and M2 can form VLPs together in the absence of any other viral proteins (47). Thus, M1 is an integral part of the infectious virion and plays an important role in virus assembly and budding.…”