pH-Controlled Two-Step Uncoating of Influenza Virus

Abstract

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5-6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection.

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

“…Conversely, we hypothesize that HA acylation may influence M1 layer disassembly that is necessary for fusion pore formation and expansion. M1 disassembly occurs in two independent steps, and the M1 layer must first undergo a conformational change prior to detachment from the membrane (28,29,31). We showed that the presence of other influenza virus proteins (NA, M1, and M2) diminished the impact of HA acylation on fusion pore expansion in a cell-RBC fusion assay.…”

“…Moreover, in the context of an intact virus, other factors influencing membrane fusion should be considered. The M2 ion channel plays an indirect but important role in destabilization and detachment of the matrix layer beneath the membrane, which is also an important requirement for pore enlargement (28,29). Interestingly, a recent study showed that influenza virions are not permeable to protons unless subjected to trypsin treatment (30).…”

“…Increased H ϩ and K ϩ concentrations (31) inside the virion interior result in weakening interactions between M1 proteins and vRNPs (32). Recent findings highlight that a gradual, rather than a steep, influenza virus acidification leads to successful vRNP delivery into the cytoplasm; this process occurs in at least two steps (29,31).…”

pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

“…11). For the limited volumes and lipid materials of an individual influenza viral particle, the acidification of the virus interior results in (i) increased M1-lipid interaction (28) and (ii) M1-M1 repulsion leading to destabilization of both the protein matrix and the lipid envelope (8,11). These two electrostatic mechanisms should increase the surface area of the protein scaffold, resulting in its tighter attachment to the viral lipid envelope prior to disintegration of the M1 matrix.…”

“…A drop to pH 6.0 results in possible conformational changes in core proteins, especially M1, that prime the viral core for release from the membrane and uncoating once fusion occurs (5). This priming leads to the dissociation of M1 protein from RNP (5-7) and some softening of the viral particle (8). The conformational change in HA below pH 5.5 leads to tight contact between viral and endosomal membranes and the formation of a small fusion pore (9).…”