Influenza A Virus M1 Protein Structure Probed by In Situ Limited Proteolysis with Bromelain

Kordyukova, Larisa V.; Serebryakova, Marina V.; Polyakov, V. Y.; Ovchinnikova, T. M.; Smirnova, Yu. A.; Fedorova, N. V.; Baratova, L. A.

doi:10.2174/092986608785849254

Cited by 16 publications

(10 citation statements)

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“…Influenza virus strain A/Puerto Rico/8/34 (subtype H1N1) was propagated in 10-day-old embryonic chicken eggs and purified by centrifugation through 20% (vol/vol) sucrose in STE buffer (100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA, pH 7.4) at 21,000 rpm for 90 min at 8 °C in the SW 27.1 rotor of a Beckman-Spinco L5-75 centrifuge, as described in 33 .…”

Section: Methodsmentioning

confidence: 99%

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

Shtykova

Dadinova

Федорова

et al. 2017

Sci Rep

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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.

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Section: Methodsmentioning

confidence: 99%

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

Shtykova

Dadinova

Федорова

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Influenza virus strain A/Puerto Rico/8/34 (subtype H1N1) was propagated in 10-day-old embryonic chicken eggs and purified by centrifugation through 20% (vol/vol) sucrose in STE buffer (100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA at pH 7.4) at 21,000 rpm for 90 min at 8°C in the SW 27.1 rotor of a Beckman-Spinco L5-75 centrifuge, as described in [19].…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of Influenza A Virus Matrix Protein M1 and Its Self-Assemblies at Low pH

et al. 2013

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Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

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“…Influenza A virus strain PR/8/34 was propagated in 10-day-old embryonic chicken eggs and purified by centrifugation in 20% (vol/vol) sucrose in STE buffer (100 mM NaCl, 10 mM TrisHCl, 1 mM EDTA, pH 7.4) at 21,000 rpm for 90 min at 8°C in the SW 27.1 rotor of a Beckman-Spinco L5-75 centrifuge (29). Isolation of M1 protein from a purified virus suspension was carried out by acid-dependent solubilization of the influenza virus membrane with the mild nonionic detergent NP-40 (Igepal; Sigma) in 50 mM morpholineethanesulfonic acid (MES)-100 mM NaCl, pH 4.0, by Zhirnov's method (30).…”

Section: Purification Of M1 Proteinmentioning

confidence: 99%

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

Batishchev

Shilova

Kachala

et al. 2016

J Virol

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Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ϳ6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ϳ5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pHdependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. IMPORTANCEInfluenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coat of protein inside the viral surface reacts to the changes in acid that come soon after internalization. We found that acid makes the molecules of the protein coat push each other while they are still stuck to the virus, so that they would like to rip the membrane apart. This ripping force is known to promote membrane fusion, the process by which infection actually occurs. While it is becoming clearer that multiple processes unite to form a pathway for the entry of the influenza virus after its uptake into endosomes, it is not yet clear how and to what extent the pH-driven changes in the viral matrix contribute to that pathway. Influenza virus is an enveloped negative-strand RNA virus of the Orthomyxoviridae family (1). Its outer envelope consists of a host cell-derived bilayer lipid membrane (BLM) with the incorporated glycoproteins hemagglutinin (HA) and neuraminidase along with proton channel M2. The inner envelope of the virion is a membrane-associated scaffold of matrix protein M1, w...

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Influenza A Virus M1 Protein Structure Probed by In Situ Limited Proteolysis with Bromelain

Cited by 16 publications

References 1 publication

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

Structural Analysis of Influenza A Virus Matrix Protein M1 and Its Self-Assemblies at Low pH

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

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