tetano
Editor, Senior Moderator
J Virol. 2019 Nov 6. pii: JVI.01605-19. doi: 10.1128/JVI.01605-19. [Epub ahead of print] [h=1]Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication.[/h]
Hu B[SUP]1[/SUP], Siche S[SUP]1[/SUP], M?ller L[SUP]2[/SUP], Veit M[SUP]3[/SUP].
[h=3]Author information[/h] 1 Institut f?r Virologie, Freie Universit?t Berlin, Berlin, Germany. 2 Robert Koch-Institut, Seestr. 10, Berlin, Germany. 3 Institut f?r Virologie, Freie Universit?t Berlin, Berlin, Germany. mveit@zedat.fu-berlin.de.
[h=3]Abstract[/h] M2 of influenza virus functions as 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 vesicularisation of membranes can be performed by a variety of amphiphilic peptides we used reverse genetics to investigate whether they 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 non-natural membrane inducer RW16. Transmission EM 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 was also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication, but other helices can replace its basic (curvature-inducing) function.Importance Influenza 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 which 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.
Copyright ? 2019 American Society for Microbiology.
PMID: 31694941 DOI: 10.1128/JVI.01605-19
Hu B[SUP]1[/SUP], Siche S[SUP]1[/SUP], M?ller L[SUP]2[/SUP], Veit M[SUP]3[/SUP].
[h=3]Author information[/h] 1 Institut f?r Virologie, Freie Universit?t Berlin, Berlin, Germany. 2 Robert Koch-Institut, Seestr. 10, Berlin, Germany. 3 Institut f?r Virologie, Freie Universit?t Berlin, Berlin, Germany. mveit@zedat.fu-berlin.de.
[h=3]Abstract[/h] M2 of influenza virus functions as 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 vesicularisation of membranes can be performed by a variety of amphiphilic peptides we used reverse genetics to investigate whether they 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 non-natural membrane inducer RW16. Transmission EM 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 was also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication, but other helices can replace its basic (curvature-inducing) function.Importance Influenza 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 which 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.
Copyright ? 2019 American Society for Microbiology.
PMID: 31694941 DOI: 10.1128/JVI.01605-19