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

Shtykova, Eleonora V.; Baratova, L. A.; Федорова, Н. В.; Radyukhin, V. A.; Ksenofontov, Alexander L.; Волков, В. В.; Shishkov, A.V.; Dolgov, A. A.; Shilova, Liudmila A.; Batishchev, Oleg V.; Jeffries, Cy M.; Svergun, Dmitri I.

doi:10.1371/journal.pone.0082431

Cited by 62 publications

(79 citation statements)

References 45 publications

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“…These results suggest that ML should be a structurally polarized molecule with a stable Nterminal domain (MN) and a C-terminal domain available for proteolytic attack. The result is consistent with the previous reports about IAV M1 (Shishkov et al, 2011;Shtykova et al, 2013). Therefore, MN was further studied using X-ray crystallography method (Fig.…”

Section: Conformational Changes Between the Neutral And Acidic Thov Msupporting

confidence: 91%

pH-dependent conformational changes of a Thogoto virus matrix protein reveal mechanisms of viral assembly and uncoating

Yang

Feng

Liu

et al. 2016

Journal of General Virology

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Orthomyxoviruses are a family of ssRNA virus, including influenza virus, infectious salmon anaemia virus and Thogoto virus. The matrix proteins of orthomyxoviruses play crucial roles in some essential processes of the viral life cycle. However, the mechanisms of the matrix proteins involved in these processes remain incompletely understood. Currently, only the structure and function of the matrix protein from influenza virus have been studied. Here, we present the crystal structures of the N-terminal domain of matrix protein from Thogoto virus at pH 7.0 and 4.5. By analysing the structures, we identified the conformational changes of monomers and dimers in different pH conditions, mainly caused by two flexible loops, L3 and L5. These structural deviations would reflect the basis of viral capsid assembly or disassembly.

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Section: Conformational Changes Between the Neutral And Acidic Thov Msupporting

confidence: 91%

pH-dependent conformational changes of a Thogoto virus matrix protein reveal mechanisms of viral assembly and uncoating

Yang

Feng

Liu

et al. 2016

Journal of General Virology

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“…Our small-angle X-ray scattering experiments (18) proved that M1 is also a monomer at pH 4.7 with a small tendency to form helical oligomers but not dimers. While the C-terminal domain is most likely responsible for the interaction of M1 with viral RNP (5), there is also a possible role for this domain in protein dimerization (19).…”

mentioning

confidence: 52%

“…The deviation from equation 3 after 110 nM M1 is due not to second-layer completion (see further AFM experiments) but probably to the anisotropy of the M1 molecule (18): the C-terminal domain is responsible for M1-RNP binding, while the N-terminal domain presumably interacts with the lipid bilayer (5). Thus, different protein-protein interactions reign within the first and the second layers versus between the layers.…”

Section: M1 Adsorption At Neutral Phmentioning

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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“…Similarly, studies on FLUC-M1 also showed that membrane tubule formation requires its C domain, corroborating its essential role for FLUC-M1 polymerization (22). In vivo, the 2D M1 lattice formation is likely promoted by cell membrane, as it has been shown that FLUA-M1 and FLUC-M1 oligomer formation is strongly enhanced by membrane binding and does not require the presence of other viral proteins (22,41).…”

Section: Resultsmentioning

confidence: 63%

“…The requirement of both N and C domains for 2D lattice formation likely explains why similar 2D lattices have not been observed in the crystal structures of FLUA-M1, FLUC-M1, and THOG-M1 that only include the N domain. An earlier study using small angle X-ray scattering indicated that the full-length FLUA-M1 monomers coexist in solution with a small fraction of large clusters with a layered architecture (41), suggesting a key role of the C terminus of FLUA-M1 in the formation of 2D lattice sheets. Similarly, studies on FLUC-M1 also showed that membrane tubule formation requires its C domain, corroborating its essential role for FLUC-M1 polymerization (22).…”

Section: Resultsmentioning

confidence: 98%

The crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association

Tao¹

2017

J Immunol Tech Infect Dis

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Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.ll members of the Orthomyxoviridae family, including the influenza viruses A-D, thogotovirus, and isavirus, encode a matrix protein called M1. In the influenza A virus, M1 is produced by a colinear transcript made from the gene segment 7 (1). As one of the most abundantly made viral proteins, the influenza A virus M1 plays multiple roles during the virus life cycle. Upon viral entry, the acidification of the viral interior in the endosome weakens the interaction between M1 and the viral ribonucleoprotein complexes (vRNPs), thus allowing M1-free vRNPs to be imported to the nucleus for viral RNA replication and transcription (2, 3). As infection proceeds, newly synthesized M1 enters the nucleus to mediate the nuclear export of nascent vRNPs. A "daisy-chain" complex is formed with the vRNP binding to the C-terminal domain of M1 (4) and the N-terminal domain of M1 interacting with the nuclear export protein (NEP), also called NS2. Through its nuclear export signal (NES), NEP is specifically recognized by the cellular exportin Crm1, which then facilitates the transport of vRNPs across the nuclear membrane to the cytoplasm in a RanGTP-dependent manner (5). It has been shown that M1 binding to vRNP in the nucleus is able to block mRNA transcription (4, 6). In the cytosol, M1 interacts with the cytoplasmic tails of the glycoproteins HA and NA. Such interactions promote M1 association with lipid raft membranes and t...

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

Cited by 62 publications

References 45 publications

pH-dependent conformational changes of a Thogoto virus matrix protein reveal mechanisms of viral assembly and uncoating

pH-dependent conformational changes of a Thogoto virus matrix protein reveal mechanisms of viral assembly and uncoating

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

The crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association

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