Summary Many viruses utilize host ESCRT proteins for budding; however, influenza virus budding is thought to be ESCRT-independent. In this study we have found a role for the influenza virus M2 proton-selective ion channel protein in mediating virus budding. We observed that a highly conserved amphipathic helix located within the M2 cytoplasmic tail mediates a cholesterol-dependent alteration in membrane curvature. The 17 amino acid amphipathic helix is sufficient for budding into giant unilamellar vesicles, and mutation of this sequence inhibited budding of transfected M2 protein in vivo. We show that M2 localizes to the neck of budding virions and that mutation of the M2 amphipathic helix results in failure of the virus to undergo membrane scission and virion release. These data suggest that M2 mediates the final steps of budding for influenza viruses, bypassing the need for host ESCRT proteins.

Influenza A virus causes seasonal epidemics, sporadic pandemics and is a significant global heath burden. Influenza virus is an enveloped virus that contains a segmented negative strand RNA genome. Assembly and budding of progeny influenza virions is a complex, multistep process that occurs in lipid raft domains on the apical membrane of infected cells. The viral proteins hemagglutinin (HA) and neuraminidase (NA) are targeted to lipid rafts, causing the coalescence and enlargement of the raft domains. This clustering of HA and NA may cause a deformation of the membrane and the initiation of the virus budding event. M1 is then thought to bind to the cytoplasmic tails of HA and NA where it can then polymerize and form the interior structure of the emerging virion. M1, bound to the cytoplasmic tails of HA and NA, additionally serves as a docking site for the recruitment of the viral RNPs and may mediate the recruitment of M2 to the site of virus budding. M2 initially stabilizes the site of budding, possibly enabling the polymerization of the matrix protein and the formation of filamentous virions. Subsequently, M2 is able to alter membrane curvature at the neck of the budding virus, causing membrane scission and the release of the progeny virion. This review investigates the latest research on influenza virus budding in an attempt to provide a step-by-step analysis of the assembly and budding processes for influenza viruses.

Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral bioavailability, these cyclic CPPs should provide a powerful system for intracellular delivery of therapeutic agents and chemical probes.