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

Roberts, Kari; Leser, George P.; Ma, Chunlong; Lamb, Robert A.

doi:10.1128/jvi.01363-13

Cited by 74 publications

(108 citation statements)

References 55 publications

(79 reference statements)

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“…It was shown previously that this mutant greatly impairs virus replication (23). We showed previously that HA and M2 strongly coclustered in membranes prepared from virus-infected cells (23,28). Here the same observation was made when HA and M2 were expressed transiently (Fig.…”

Section: Resultssupporting

confidence: 81%

“…However, the pattern of coclustering of HA and M2 may reflect the role that M2 is believed to play in scission; by occupying the periphery of HA patches, M2 is positioned to concentrate at the base of nascent budding virions (70). The amphipathic helix in the cytoplasmic tail of M2 has been shown to modify membrane curvature and is necessary for efficient membrane scission and virus release (28,(70)(71)(72). When the distribution of HA or NA is compared to that of a mutant M2 protein with cytoplasmic tail residues 71 to 73 replaced with alanine, a mutation known to impair virus growth, likely by interrupting the interaction of M2 with M1 (23), the magnitude of coclustering is dramatically reduced, suggesting a direct or indirect impact on HA or NA interactions ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Residues 45 to 62 of the M2 cytoplasmic tail form an amphipathic helix, which has been implicated in playing a role in the budding process by inducing membrane curvature (26). Mutation of the amphipathic helix of M2 results in altered budding, as viral filament formation is impaired (25,27,28). A functional link between M1 and M2 was demonstrated when escape mutants of a monoclonal antibody (14C2) specific for the M2 ectodomain were found to restrict virus growth and were mapped to the M1 protein (29).…”

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confidence: 99%

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Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

Leser

Lamb

2017

J Virol

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Influenza virus assembles and buds at the plasma membrane of virusinfected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

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Section: Resultssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

Leser

Lamb

2017

J Virol

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“…The C-terminal amphipathic helix of M2 is essential for endosomal sorting complex required for transcription (ESCRT)-independent virus scission and filamentous virus formation (40), acting to stabilize the negative Gaussian curvature associated with the budding intermediate (41). Although the sequence and amphipathic character of this helix are essential to these processes, mutations that affect these parameters do not have a measurable effect on the conductance of the channel (41).…”

Section: Significancementioning

confidence: 99%

Acid activation mechanism of the influenza A M2 proton channel

Liang

Swanson

Madsen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

146

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The homotetrameric influenza A M2 channel (AM2) is an acidactivated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.ion channel | proton conduction | multiscale modeling | QM/MM | free-energy sampling V isualizing the vectorial flow of protons through membrane proteins is a long-standing challenge in biophysics and chemistry, with manifold implications for understanding bioenergetics, active transport, and proton channel function. Multiscale modeling has become a full partner with experimental structural biology in addressing proton transport (PT), because it can connect the dots between the high-resolution but static pictures obtained from crystallography and the lower-resolution but dynamically informed structures seen in the ensemble averages by NMR and other spectroscopic approaches. In general, multiscale modeling connects three or more disparate but coupled scales of behavior. In the case of PT in proteins, there are usually four pertinent scales: (i) the quantum mechanical scale of bond breaking and bond making inherent in the Grotthuss proton shuttle mechanism; (ii) the molecular scale of the dynamical motions of the protein, membrane, ions, and water molecules; (iii) the "free energy" scale that arises from the statistical ensemble averaging of those molecular motions; and (iv) the "transport" scale that manifests as a macroscopic conductance, with associated gating and other possible behaviors tied to macroscopic experimental variables. The proper and rigorous connection of these various scales, in an overall multiscale computational simulation, is often a substantial challenge. Here, we turn our attention to understanding the me...

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“…The regions C-terminal to the TM helices play different important functional roles. An amphiphilic helix formed by residues 46-60 induces membrane curvature and is involved in viral budding and scission (21)(22)(23), and an intrinsically disordered C-terminal tail (residues 62-97) interacts with the matrix 1 protein during the packing and budding of new virus particles (24,25).…”

Section: Significancementioning

confidence: 99%

XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction

Thomaston

Woldeyes

Nakane

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inward open state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inward open state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.W ater molecules in transmembrane protein pores participate in the transport of protons across the membrane bilayer. This process has been extensively studied experimentally and through computational simulations, particularly in small channels such as gramicidin A and influenza A M2. The movement of ions through channels is coupled to the diffusion of water through the pore, but protons are transported at a rate that is faster than the diffusion of H 3 O + (1, 2). Instead of diffusing through channels, protons move concertedly across networks of hydrogen-bonded waters through what is known as the Grotthuss mechanism (3-5). This mechanism of proton transport was initially discovered by the behavior of water in solution, and it has also been proposed to occur within membrane proteins containing water-filled pores (6-9). The matrix 2 (M2) protein of influenza A is one of the smallest proton-selective channels found in nature. This makes it an ideal system for studying the involvement of water in the selective transport of protons across the membrane. The M2 protein of influenza A is a tetramer made up of four 97-residue-long monomers. M2 is multifunctional, with different functions lying in different regions of the sequence. Residues 1-22 make up a conserved N-terminal domain that assists the incorporation of M2 into the virion (10) and is absent in influenza B viruses. The transmembrane domain of M2 (residues 22-46) is necessary for tetramerization (11) and forms a proton-selective channel (12-15) that is the target of the adamantane class of drugs, amantadine and rimantadine (11,(16)(17)(18). The transme...

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

Cited by 74 publications

References 55 publications

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

Acid activation mechanism of the influenza A M2 proton channel

XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction

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