N-terminal α-helix-independent membrane interactions facilitate adenovirus protein VI induction of membrane tubule formation

Abstract

Adenovirus disrupts endosomal membranes during cell entry. The membrane lytic capsid protein VI (pVI) facilitates entry by fragmenting membranes. Although an N-terminal amphipathic α-helix (VI-Φ) possesses similar membrane affinity as pVI, truncated protein lacking VI-Φ, (VIΔ54) still possesses moderate membrane affinity. We demonstrate that incorporation of nickel-NTA lipids in membranes enhances increases the membrane affinity and the membrane lytic activity of VIΔ54. We also demonstrate that 3 predicted pVI α-helices within residues 54–114 associate with membranes, sitting roughly parallel to the membrane surface. His-tagged VIΔ54 is capable of fragmenting membranes similar to pVI and the VI-Φ peptide. Interestingly, neither VI-Φ nor His-tagged VIΔ54 can induce tubule formation in giant lipid vesicles as observed for pVI. These data suggest cooperativity between the amphipathic α-helix and residues in VIΔ54 to induce positive membrane curvature and tubule formation. These results provide additional details regarding the mechanism of nonenveloped virus membrane penetration.

A Single Maturation Cleavage Site in Adenovirus Impacts Cell Entry and Capsid Assembly

“…In further studies, we sought to determine the functional consequences of hexon-pVIn association. The membrane lytic activity of protein VI primarily lies within the N-terminal amphipathic helix (residues 34 to 54) of mature VI (7,25). When we compared recombinant pVI with VI in an in vitro fluorescence-based liposome lysis assay, we observed very similar dose-dependent lytic activities for the two forms of the protein (Fig.…”

Viral weapons of membrane destruction: variable modes of membrane penetration by non-enveloped viruses

“…Recent studies also suggest that the hydrophobic face of the amphipathic helix of protein VI inserts in a parallel fashion into the lipid bilayer, with the hydrophilic face contacting the phospholipid head groups [37]. Furthermore, membrane fragmentation may occur via induction of positive curvature, as suggested by experiments assessing membrane disruption of giant unilamellar vesicles by protein VI [37, 38]. This in vitro evidence suggests that protein VI mediates endosome lysis by imparting stress on the luminal leaflet of the endosomal membrane (Figure 1).…”

“…AdV protein VI inserts into the lumenal leaflet of the endosomal membrane, inducing positive membrane curvature and total fragmentation of the endosome. This model is based on VI-mediated lysis of unilamellar vesicles [37, 38]. Viruses are scaled to depict the relative size of each particle.…”

Adenovirus membrane penetration: Tickling the tail of a sleeping dragon

“…When this N-terminal amphipathic α-helix is replaced by a 6xHis tag the membrane binding capacity is reduced 40-fold and membrane lytic activity is reduced by ∼100-fold (56). However, if membrane affinity of this 6xHis-tag-replaced protein VI is artificially enhanced by using liposomes containing NTA-Ni2+ headgroups for binding to the 6xHis tag, then the membrane lytic activity of the protein can be restored to normal levels (58). Further genetic evidence that this N-terminal amphipathic α-helix is required for membrane binding and rupture was obtained when L40 was mutated to glutamine, L40Q (57).…”

“…Within seconds after addition of protein VI, these lipid vesicles are completely fragmented. In many cases, these fragments are rearranged into elongated tubules (58), although the limits of light microscopy spatial resolution has precluded further insights into the structure of these tubules. Nonetheless, these observations fit with a model in which protein VI binds membranes and, much like a detergent, fragments the lipid bilayer into smaller, highly curved structures.…”