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

Abstract

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.

A Thermal-Stable Protein Nanoparticle That Stimulates Long Lasting Humoral Immune Response

“…It mediates virus budding by generating membrane curvature by embedding its hydrophobic face in the membrane bilayer [ 30 ]. Additionally, it anchors the M2 ectopic domain on the viral envelope, which serves as a proton pump that triggers envelope membrane fusion during infection [ 41 ]. Here we discovered a novel application of AH3, which serves as a nucleating center for protein nanoparticle assembly.…”

Functions of Viroporins in the Viral Life Cycle and Their Regulation of Host Cell Responses

“…For enveloped viruses, budding and division are typically used to release virus from infected cells, and the mechanisms involved in the release process vary from virus to virus. During influenza virus release, the M2 protein is located at the neck of the budding virion, and the amphiphilic helix inserts into the membrane, inducing bending of the positive membrane, and finally pinching off the budding virion ( 218 , 222 ). Mutations or deletions in the M2 cytoplasmic tail structural domain (CTD) significantly impair the assembly of viral proteins and genomic fragments into viral particles and viral particle release ( 218 , 223 , 224 ).…”

Vemurafenib Inhibits Enterovirus A71 Genome Replication and Virus Assembly

“…Culture media was harvested when over 50% cells showed CPE. After clearing cell debris by low-speed centrifugation (4000 g, 5 min), viruses were pelleted by ultracentrifugation (175,000g, 1.5h) through 20% sucrose cushion and resuspended in 1X TNE buffer (10 mM Tris, 100 mM NaCl und 1 mM EDTA, pH 7.4) as previously described [37,38], and then subjected to western blot analysis.…”