Abstract
Influenza A virus matrix protein M1 plays an essential role in the virus lifecycle, but its functional and structural properties are not entirely defined. Here we employed small-angle X-ray scattering, atomic force microscopy and zeta-potential measurements to characterize the overall structure and association behavior of the full-length M1 at different pH conditions. We demonstrate that the protein consists of a globular N-terminal domain and a flexible C-terminal extension. The globular N-terminal domain of M1 monomers appears preserved in the range of pH from 4.0 to 6.8, while the C-terminal domain remains flexible and the tendency to form multimers changes dramatically. We found that the protein multimerization process is reversible, whereby the binding between M1 molecules starts to break around pH 6. A predicted electrostatic model of M1 self-assembly at different pH revealed a good agreement with zeta-potential measurements, allowing one to assess the role of M1 domains in M1-M1 and M1-lipid interactions. Together with the protein sequence analysis, these results provide insights into the mechanism of M1 scaffold formation and the major role of the flexible and disordered C-terminal domain in this process.
Highlights
Influenza viruses belongs to the most widespread and potentially dangerous pathogenic group of viruses in the world[1]
Solution studies of the full-length protein using dynamic light scattering and small-angle neutron scattering indicated that M1 adopts an elongated monomeric form[18] at neutral pH, while our previous small angle X-ray scattering experiments[14] showed full-length M1 at pH 4.7 is predominantly monomeric with small fraction of the protein in the form of helical oligomers, close to those observed in intact viral particles at neutral pH20,21
These results suggest that electrostatic forces are a main contributor to M1-M1 and M1-lipid interactions and their modulation with pH, some charge-independent forces are responsible for the interaction of the protein with itself and with lipid membranes
Summary
Influenza viruses belongs to the most widespread and potentially dangerous pathogenic group of viruses in the world[1]. A pH drop inside the late endosome triggers the fusion of the viral and endosomal membranes[5] Under these conditions, the first step is a priming stage[6] at a pH of around 6.0 that causes a possible conformational change in M1 protein and the subsequent dissociation from the viral RNP6–8 resulting in a loss of viral particle rigidity[9]. Further decreases in pH results in the acidification of the medium inside the viral particle due to the action of proton channels M2 This process leads to a disintegration of the viral protein scaffold resulting from the increased charge of M1 monomers in acidic medium[10], and their partial dissociation from the viral lipid envelope[13]. We suggest a model of pH-dependent oligomerization of M1 and calculate the energy of the interaction of the individual M1 molecules as well as an estimate of M1 protein charge at different values of pH
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