Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering

Curthoys, Nikki M.; Mlodzianoski, Michael J.; Parent, Matthew; Butler, Michael B.; Raut, Prakash; Wallace, Jaqulin N.; Lilieholm, Jennifer; Mehmood, Kashif; Maginnis, Melissa S.; Waters, Hang; Busse, Brad; Zimmerberg, Joshua; Hess, Samuel T.

doi:10.1016/j.bpj.2019.01.017

Cited by 37 publications

(82 citation statements)

References 85 publications

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“…Whether M2 ALPS and M2 Epsin are enriched at the viral budding site cannot be concluded from our data. Note, however, that the ␣0-helix of Epsin binds to PtdIns(4,5)P2 even outside the ETNH domain and that HA also colocalizes with PtdIns(4,5)P2 (65). Thus, the proposed mutual affinity of HA and M2 Epsin for this negatively charged lipid present only at the inner leaflet of the plasma membrane might cause their colocalization.…”

Section: Discussionmentioning

confidence: 97%

Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication

Siche

Møller

et al. 2020

J Virol

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M2 of influenza virus functions as a proton channel during virus entry. In addition, an amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the assembly site where it inserts into the inner membrane leaflet to induce curvature that causes virus scission. Since vesicularization of membranes can be performed by a variety of amphiphilic peptides, we used reverse genetics to investigate whether the peptides can substitute for M2’s helix. Virus could not be generated if M2’s helix was deleted or replaced by a peptide predicted not to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was exchanged by helices known to induce membrane curvature. Infectious virus titers were marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor from the Epsin N-terminal homology domain or the nonnatural membrane inducer RW16. Transmission electron microscopy of infected cells did not reveal unequivocal evidence that virus budding or membrane scission was disturbed in any of the mutants. Instead, individual virus mutants exhibit other defects in M2, such as reduced surface expression, incorporation into virus particles, and ion channel activity. The protein composition and specific infectivity were also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication but that other helices can replace its basic (curvature-inducing) function. IMPORTANCE Influenza virus is unique among enveloped viruses since it does not rely on the cellular ESCRT machinery for budding. Instead, viruses encode their own scission machine, the M2 protein. M2 is targeted to the edge of the viral assembly site, where it inserts an amphiphilic helix into the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins with only small effects on virus replication. No evidence was obtained that budding is disturbed, but individual mutants exhibit other defects in M2 that explain the reduced virus titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic helix to induce membrane curvature, but its biophysical properties are more important than the amino acid sequence.

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Section: Discussionmentioning

confidence: 97%

Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication

Siche

Møller

et al. 2020

J Virol

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show abstract

“…Whether M2 ALPS and M2 Epsin are enriched at the viral budding site cannot be concluded from our data. Note, however, that the α0-helix of Epsin binds to PtdIns(4, 5)P2 even outside of the ETNH-domain and that HA also co-localizes with PtdIns(4, 5)P2 (65). Thus, the proposed mutual affinity of HA and M2 Epsin for this negatively charged lipid present only at the inner leaflet of the plasma membrane might cause their co-localization.…”

Section: Discussionmentioning

confidence: 99%

Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication

Siche

Møller

et al. 2019

Preprint

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M2 of influenza virus functions as proton channel during virus entry. In addition, an 1 amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the 2 assembly site where it inserts into the inner membrane leaflet to induce curvature that causes 3 virus scission. Since vesicularisation of membranes can be performed by a variety of 4 amphiphilic peptides we used reverse genetics to investigate whether they can substitute for 5 M2´s helix. 6 Virus could not be generated if M2´s helix was deleted or replaced by a peptide predicted not 7 to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was 8 exchanged by helices known to induce membrane curvature. Infectious virus titers were 9 marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor, from the 10 Epsin N-Terminal Homology domain or the non-natural membrane inducer RW16. 11Transmission EM of infected cells did not reveal unequivocal evidence that virus budding or 12 membrane scission was disturbed in any of the mutants. Instead, individual virus mutants 13 exhibit other defects in M2, such as reduced surface expression, incorporation into virus 14 particles and ion channel activity. The protein composition and specific infectivity was also 15 altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is 16 essential for virus replication, but other helices can replace its basic (curvature-inducing) 17 function. 18 3 Importance 19Influenza is unique among enveloped viruses since it does not rely on the cellular ESCRT-20 machinery for budding. Instead viruses encode their own scission machine, the M2 protein. 21M2 is targeted to the edge of the viral assembly site where it inserts an amphiphilic helix into 22 the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission 23 of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins 24 with only small effects on virus replication. No evidence was obtained that budding is 25 disturbed, but individual mutants exhibit other defects in M2 which explain the reduced virus 26 titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by 27 irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic 28 helix to induce membrane curvature, but its biophysical properties are more important than 29 the amino acid sequence. 30 4 71membrane curvature as shown by preferential binding of M2 to small unilamellar vesicles 72 (SUVs) having a small diameter and by molecular dynamics simulations (31, 32). More 73 specifically, clusters of M2 molecules are excluded from regions with negative curvature, i. e. 74 the outward part of a budding virus particle, but rather accumulate at membrane regions with 75 positive curvature, i. e. the neck of a budding virus (33). 76Positioning of M2 at the edge of the viral budding zone could then entail the scission of virus 77 particles: the amphiphilic helix...

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“…Depletion of the second late domain motif PPRY also reduces virus shedding, with the majority of particles remaining outside intracellular vesicles rather than inside for the WT BTV particles [ 50 ]. Another striking similarity between BTV and enveloped virus maturation is the involvement of the lipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2), which is a key effector of virus release [ 51 , 52 ]. It has been reported that PI(4,5)P2 co-localises with NS3 and VP5, and that depletion of this lipid reduced BTV maturation and release [ 53 ].…”

Section: The Membrane Protein Ns3 Is Responsible For Trafficking Amentioning

confidence: 99%

Multiple Routes of Bluetongue Virus Egress

2020

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Bluetongue virus (BTV) is an arthropod-borne virus infecting livestock. Its frequent emergence in Europe and North America had caused significant agricultural and economic loss. BTV is also of scientific interest as a model to understand the mechanisms underlying non-enveloped virus release from mammalian and insect cells. The BTV particle, which is formed of a complex double-layered capsid, was first considered as a lytic virus that needs to lyse the infected cells for cell to cell transmission. In the last decade, however, a more in-depth focus on the role of the non-structural proteins has led to several examples where BTV particles are also released through different budding mechanisms at the plasma membrane. It is now clear that the non-structural protein NS3 is the main driver of BTV release, via different interactions with both viral and cellular proteins of the cell sorting and exocytosis pathway. In this review, we discuss the most recent advances in the molecular biology of BTV egress and compare the mechanisms that lead to lytic or non-lytic BTV release.

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Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering

Cited by 37 publications

References 85 publications

Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication

Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication

Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication

Multiple Routes of Bluetongue Virus Egress

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