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

Shtykova, Eleonora V.; Dadinova, L. A.; Федорова, Н. В.; Golanikov, A. E.; Bogacheva, E. N.; Ksenofontov, Alexander L.; Baratova, L. A.; Shilova, Liudmila A.; Tashkin, V. Yu.; Galimzyanov, Timur R.; Jeffries, Cy M.; Svergun, Dmitri I.; Batishchev, Oleg V.

doi:10.1038/s41598-017-16986-y

Cited by 27 publications

(36 citation statements)

References 72 publications

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“…It is worth noting that the M1 structure described here is to be considered as one of several possible conformations that the protein can adopt. This assumption is clearly corroborated by the similarity between the M1 structural model presented here and one of several possible M1 structures that were recently described [64]. Our model of M1 in solution was then used to analyze M1–membrane interaction by MDS and to provide an interpretation of our experimental data on a molecular and atomistic level.…”

Section: Discussionsupporting

confidence: 83%

“…The fact that no significant structural rearrangements are observed for a single membrane-bound M1 molecule by MDS, suggests that the changes observed in the CD spectra of full-length M1 might be caused by protein–protein interactions, occurring concurrently to protein–lipid interactions. The structural plasticity of M1 as previously discussed [36, 37, 64] and, in particular, conformational changes in the full-length protein as we report here (especially in the C-terminal domain) might be required for the multi-functionality of M1, including its ability to multimerize and mediate diverse steps in the infection process.…”

Section: Discussionsupporting

confidence: 59%

“…Small-angle neutron scattering [33] and a recent low-resolution reconstruction of the full-length M1 from small-angle X-ray scattering suggest that the protein has an elongated shape with a globular N-terminal domain and an elongated and flexible C-terminal domain [36]. Finally, very recent small-angle X-ray scattering (including ROSETTA modelling and docking analysis) confirmed these general findings and proposed the existence of multiple structural configurations that the flexible C-terminal domain can adopt [64].…”

Section: Discussionmentioning

confidence: 93%

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Structural Determinants of the Interactions Between Influenza A Virus Matrix Protein M1 and Lipid Membranes

Hoefer

Lella

Dahmani

et al. 2018

Preprint

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13 Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human 14 and animal populations every year. One of the ten proteins encoded by the viral genome, the 15 matrix protein M1, is abundantly produced in infected cells and plays a structural role in 16 determining the morphology of the virus. During assembly of new viral particles, M1 is 17recruited to the host cell membrane where it associates with lipids and other viral proteins. 18The structure of M1 is only partially known. In particular, structural details of M1 interactions 19with the cellular plasma membrane as well as M1-protein interactions and multimerization 20have not been clarified, yet. 21In this work, we employed a set of complementary experimental and theoretical tools to 22 tackle these issues. Using raster image correlation, surface plasmon resonance and circular 23 dichroism spectroscopies, we quantified membrane association and oligomerization of full-24 length M1 and of different genetically engineered M1 constructs (i.e., N-and C-terminally 25 truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we 26 report novel information on structural changes in M1 occurring upon binding to membranes. 27 Our experimental results are corroborated by an all-atom model of the full-length M1 protein 28 bound to a negatively charged lipid bilayer. 29 30 31 Influenza A viruses (IAV) circulate in human and animal populations, posing a significant 32 threat to global health. They are responsible for severe pathologies with high mortality during 33 seasonal epidemics and occasional pandemic outbreaks [1]. IAVs belong to the family of 34 Orthomyxoviridae and are negative-sense single-stranded RNA viruses [2]. The segmented 35 viral genome, in form of eight viral ribonucleoprotein complexes (vRNPs), is enclosed by the 36 viral lipid envelope [3]. The matrix layer underneath the viral membrane is formed by the 37 matrix protein M1, which consists of 252 amino acid (aa) residues [4-8]. M1 is the most 38 abundant protein in virus particles, stabilizes the virion structure and is considered to be a 39 determinant of viral morphology [9-13]. Electron micrographs demonstrate tight association 40 of the matrix layer with the viral lipid membrane [5]. M1 is abundantly produced in infected 41 cells and distributes throughout cytoplasm and nuclear compartment. In addition to its role as 42 a structural protein, M1 has other functions during the viral replication cycle. It is involved in 43 nucleocytoplasmic transport of the viral genome and, therefore, plays a role in coordinating 44 nuclear-localized replication and assembly of progeny viruses at the plasma membrane (PM) 45 [14-19]. M1 is furthermore considered to be a key player during virus assembly because of its 46 ability to associate with all other structural components of virus particles, including the 47 genomic vRNP complexes [20-22], the three viral transmembrane proteins (i.e. hemagglutinin 48 (HA), neuraminidase (NA) and the proton chan...

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Section: Discussionsupporting

confidence: 83%

Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 93%

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Structural Determinants of the Interactions Between Influenza A Virus Matrix Protein M1 and Lipid Membranes

Hoefer

Lella

Dahmani

et al. 2018

Preprint

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“…Second, we incubated GUVs containing DOPC and 40 mol% DOPS in the presence of 10 µM M1-Alexa488 at pH 5. Low pH was already shown to interfere with M1 multimerization (7,(32)(33)(34)(35)(36)(37). Once again, we did not observe significant alterations in the shape of the GUVs (see e.g.…”

Section: M1-m1 Interactions Are Needed For Membrane Deformationsupporting

confidence: 67%

Influenza A matrix protein M1 is sufficient to induce lipid membrane deformation

Dahmani

Ludwig

Chiantia

2019

Preprint

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The matrix protein M1 of the Influenza A virus is considered to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle towards the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In this study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed capable to cause membrane curvature in lipid bilayers containing negatively-charged lipids, in the absence of other viral components. Furthermore, we prove that simple protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1-M1 interactions and multimer formation are required in order to alter the bilayer threedimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context, M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains). FIGURE 4: M1 layer interacting with the membrane is stable even after lipid removal. A-B: LSM confocal image of a typical GUV composed of DOPC:cholesterol:DOPS 50:20:30, after incubation with 5 µM M1-Alexa488 (green channel, panel A). The lipid bilayer is visualized via the addition of 0.05 mol% Rhodamine-DOPE (red channel, panel B). C-D: LSM confocal image of a typical GUV after treatment for 5 min with detergent (e.g. Triton X-100 1.7 mM). Panel C represent the signal from M1-Alexa488. Panel D represents the signal from Rhodamine-DOPE. The excitation laser power used to acquire the image shown in panel D was ~10 times higher than the power used to acquire the image shown in panel B. GUVs contained 150 mM sucrose in their lumen and were suspended in a phosphate buffered protein solution (pH 7.4) with similar osmolarity (see Materials and Methods). Scale bars are 5 µM. Images were acquired at 23°C.

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“…7b, curve 1). The resulting SAXS profile was used to reconstruct the shape of the M-NDV clusters similar to the approach used in Shtykova et al (32,33).…”

Section: Saxs Measurements Of the M-ndv Protein In Solutionmentioning

confidence: 99%

Solution Structure, Self-Assembly, and Membrane Interactions of the Matrix Protein from Newcastle Disease Virus at Neutral and Acidic pH

Shtykova

Petoukhov

Dadinova

et al. 2019

J Virol

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Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration. IMPORTANCE The matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to the Avulavirus subfamily of the Paramyxoviridae family of Mononegavirales that include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here, we investigate the solution structure and membrane binding properties of this protein at both acidic and neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals for the possible use of the virus as an adequate template for future drug or vaccine delivery. KEYWORDS Newcastle disease virus, ab initio modeling, atomic force microscopy, matrix protein, protein structure, small-angle X-ray scattering Citation Shtykova EV, Petoukhov MV, RESULTSCD analysis of the isolated M-NDV protein.To characterize the secondary structure of the isolated matrix protein, we performed circular dichroism (CD) spectropolarimetry measurements. In the present work, we used a Chirascan dichrograph to measure the far-UV CD spectrum of isolated M-NDV in 50 mM morpholineethanesulfonic acid (MES) and 100 mM NaCl buffer, pH 4.0 and 7.0 ( Fig. 1). In the...

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

Cited by 27 publications

References 72 publications

Structural Determinants of the Interactions Between Influenza A Virus Matrix Protein M1 and Lipid Membranes

Structural Determinants of the Interactions Between Influenza A Virus Matrix Protein M1 and Lipid Membranes

Influenza A matrix protein M1 is sufficient to induce lipid membrane deformation

Solution Structure, Self-Assembly, and Membrane Interactions of the Matrix Protein from Newcastle Disease Virus at Neutral and Acidic pH

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