Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Lipid-specific oligomerization of the Marburg virus matrix protein VP40 is regulated by two distinct interfaces for virion assembly

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100796

ISSN

1083-351X

Autores

Souad Amiar, Monica L. Husby, Kaveesha J. Wijesinghe, Stephanie Angel, Nisha Bhattarai, Bernard S. Gerstman, Prem P. Chapagain, Sheng Li, Robert V. Stahelin,

Tópico(s)

Hepatitis B Virus Studies

Resumo

Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD–NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD–CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein–protein or lipid–protein interactions to inhibit virus budding. Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD–NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD–CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein–protein or lipid–protein interactions to inhibit virus budding. The Filoviridae family of viruses, which includes Marburg virus (MARV) and its cousin Ebola virus (EBOV), has been responsible for several highly fatal outbreaks since the late 1960s (1Leroy E.M. Gonzalez J.-P. Baize S. Ebola and Marburg haemorrhagic fever viruses: Major scientific advances, but a relatively minor public health threat for Africa.Clin. Microbiol. Infect. 2011; 17: 964-976Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 2World Health OrganizationEbola Virus Disease: Democratic Republic of the Congo. Elsevier, Amsterdam, Netherlands2019Google Scholar, 3Suzuki Y. Gojobori T. The origin and evolution of Ebola and Marburg viruses.Mol. Biol. Evol. 1997; 4: 800-806Crossref Scopus (78) Google Scholar, 4Breman J.G. Heymann D.L. Lloyd G. McCormick J.B. Miatudila M. Murphy F.A. Muyembé-Tamfun J.-J. Piot P. Ruppol J.-F. Sureau P. van der Groen G. Johnson K.M. Discovery and description of Ebola Zaire virus in 1976 and relevance to the west African epidemic during 2013–2016.J. Infect. Dis. 2016; 214: S93-S101Crossref PubMed Scopus (36) Google Scholar, 5Slenczka W. Klenk H.D. Forty years of Marburg virus.J. Infect. Dis. 2007; 196: S131-S135Crossref PubMed Scopus (56) Google Scholar). Filoviruses are lipid-enveloped viruses harboring a negative-sense RNA genome, which bud and release new filamentous viral particles from the host cell plasma membrane (1Leroy E.M. Gonzalez J.-P. Baize S. Ebola and Marburg haemorrhagic fever viruses: Major scientific advances, but a relatively minor public health threat for Africa.Clin. Microbiol. Infect. 2011; 17: 964-976Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 6Beer B. Kurth R. Bukreyev A. Characteristics of Filoviridae: Marburg and Ebola viruses.Naturwissenschaften. 1999; 86: 8-17Crossref PubMed Scopus (50) Google Scholar, 7Noda T. Sagara H. Suzuki E. Takada A. Kida H. Kawaoka Y. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP.J. Virol. 2002; 76: 4855-4865Crossref PubMed Scopus (271) Google Scholar, 8Bray M. Geisbert T.W. Ebola virus: The role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever.Int. J. Biochem. Cell Biol. 2005; 37: 1560-1566Crossref PubMed Scopus (203) Google Scholar, 9Kolesnikova L. Bugany H. Klenk H. Becker S. VP40, the matrix protein of Marburg virus, is associated with membranes of the late endosomal compartment.J. Virol. 2002; 76: 1825-1838Crossref PubMed Scopus (84) Google Scholar, 10Kolesnikova L. Bamberg S. Berghofer B. Becker S. The matrix protein of Marburg virus is transported to the plasma membrane along cellular membranes: Exploiting the retrograde late endosomal pathway.J. Virol. 2004; 78: 2382-2393Crossref PubMed Scopus (65) Google Scholar, 11Bharat T.A.M. Noda T. Riches J.D. Kraehling V. Kolesnikova L. Becker S. Kawaoka Y. Briggs J.A.G. Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 4275-4280Crossref PubMed Scopus (143) Google Scholar). The viral matrix protein VP40 of MARV and EBOV (mVP40 and eVP40, respectively) is the primary viral component responsible for directing the assembly and budding of viral particles from the host cell plasma membrane inner leaflet (7Noda T. Sagara H. Suzuki E. Takada A. Kida H. Kawaoka Y. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP.J. Virol. 2002; 76: 4855-4865Crossref PubMed Scopus (271) Google Scholar, 9Kolesnikova L. Bugany H. Klenk H. Becker S. VP40, the matrix protein of Marburg virus, is associated with membranes of the late endosomal compartment.J. Virol. 2002; 76: 1825-1838Crossref PubMed Scopus (84) Google Scholar, 12Feldmann H. Klenk H.D. Sanchez A. Molecular biology and evolution of filoviruses.Arch. Virol. Suppl. 1993; 7: 81-100Crossref PubMed Scopus (128) Google Scholar, 13Harty R.N. Brown M.E. Wang G. Huibregtse J. Hayes F.P. A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: Implications for filovirus budding.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13871-13876Crossref PubMed Scopus (370) Google Scholar, 14Jasenosky L.D. Neumann G. Lukashevich I. Kawaoka Y. Ebola virus VP40-induced particle formation and association with the lipid bilayer.J. Virol. 2001; 75: 5205-5214Crossref PubMed Scopus (227) Google Scholar). Indeed, VP40 expression alone is sufficient to produce virus-like particles (VLPs) with similar morphology to virions when expressed in mammalian cells (7Noda T. Sagara H. Suzuki E. Takada A. Kida H. Kawaoka Y. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP.J. Virol. 2002; 76: 4855-4865Crossref PubMed Scopus (271) Google Scholar, 9Kolesnikova L. Bugany H. Klenk H. Becker S. VP40, the matrix protein of Marburg virus, is associated with membranes of the late endosomal compartment.J. Virol. 2002; 76: 1825-1838Crossref PubMed Scopus (84) Google Scholar, 13Harty R.N. Brown M.E. Wang G. Huibregtse J. Hayes F.P. A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: Implications for filovirus budding.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13871-13876Crossref PubMed Scopus (370) Google Scholar, 14Jasenosky L.D. Neumann G. Lukashevich I. Kawaoka Y. Ebola virus VP40-induced particle formation and association with the lipid bilayer.J. Virol. 2001; 75: 5205-5214Crossref PubMed Scopus (227) Google Scholar). Understanding the mechanism by which filoviruses assemble to form new virions is tightly related to understanding VP40 structure–function properties upon protein binding to target lipid that may induce or stabilize VP40 oligomers. VP40 is a peripheral protein, and mVP40 lipid binding was first speculated when the protein was shown to accumulate at intracellular membranes, mostly multivesicular bodies and late endosomes early after its synthesis in cells (9Kolesnikova L. Bugany H. Klenk H. Becker S. VP40, the matrix protein of Marburg virus, is associated with membranes of the late endosomal compartment.J. Virol. 2002; 76: 1825-1838Crossref PubMed Scopus (84) Google Scholar, 10Kolesnikova L. Bamberg S. Berghofer B. Becker S. The matrix protein of Marburg virus is transported to the plasma membrane along cellular membranes: Exploiting the retrograde late endosomal pathway.J. Virol. 2004; 78: 2382-2393Crossref PubMed Scopus (65) Google Scholar, 15Kolesnikova L. Berghofer B. Bamberg S. Becker S. Multivesicular bodies as a platform for formation of the Marburg virus envelope.J. Virol. 2004; 78: 12277-12287Crossref PubMed Scopus (89) Google Scholar). Later, the critical role of anionic lipids, phosphatidylserine (PS) and phosphoinositides (PIPs), for both mVP40 and eVP40 trafficking and interactions with the plasma membrane inner leaflet has been more well established (16Oda S. Noda T. Wijesinghe K.J. Halfmann P. Bornholdt Z.A. Abelson D.M. Armbrust T. Stahelin R.V. Kawaoka Y. Saphire E.O. Crystal structure of Marburg virus VP40 reveals a broad, basic patch for matrix assembly and a requirement of the N-terminal domain for immunosuppression.J. Virol. 2016; 90: 1839-1848Crossref PubMed Scopus (26) Google Scholar, 17Wijesinghe K.J. Stahelin V. Investigation of the lipid binding properties of the Marburg virus matrix protein VP40.J. Virol. 2016; 90: 3074-3085Crossref Scopus (17) Google Scholar, 18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 19Ruigrok R.W.H. Schoehn G. Dessen A. Forest E. Volchkov V. Dolnik O. Klenk H.D. Weissenhorn W. Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.J. Mol. Biol. 2000; 300: 103-112Crossref PubMed Scopus (124) Google Scholar, 20Adu-Gyamfi E. Soni S.P. Xue Y. Digman M.A. Gratton E. Stahelin R.V. The Ebola virus matrix protein penetrates into the plasma membrane: A key step in viral protein 40 (VP40) oligomerization and viral egress.J. Biol. Chem. 2013; 288: 5779-5789Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Adu-Gyamfi E. Johnson K.A. Fraser M.E. Scott J.L. Soni S.P. Jones K.R. Digman M.A. Gratton E. Tessier C.R. Stahelin R.V. Host cell plasma membrane phosphatidylserine regulates the assembly and budding of Ebola virus.J. Virol. 2015; 89: 9440-9453Crossref PubMed Scopus (55) Google Scholar, 22Johnson K.A. Taghon G.J.F. Scott J.L. Stahelin R.V. The Ebola virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2) for extensive oligomerization at the plasma membrane and viral egress.Sci. Rep. 2016; 12: 19125Crossref Scopus (45) Google Scholar). VP40 forms a dimer (16Oda S. Noda T. Wijesinghe K.J. Halfmann P. Bornholdt Z.A. Abelson D.M. Armbrust T. Stahelin R.V. Kawaoka Y. Saphire E.O. Crystal structure of Marburg virus VP40 reveals a broad, basic patch for matrix assembly and a requirement of the N-terminal domain for immunosuppression.J. Virol. 2016; 90: 1839-1848Crossref PubMed Scopus (26) Google Scholar, 23Bornholdt Z.A. Noda T. Abelson D.M. Halfmann P. Wood M.R. Kawaoka Y. Saphire E.O. Structural rearrangement of Ebola virus VP40 begets multiple functions in the virus life cycle.Cell. 2013; 154: 763-774Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) involving interaction at the amino-terminal domain (NTD). In addition, this NTD is involved in large oligomer formation. The carboxy-terminal domain (CTD) containing the lipid-binding loops is important for VP40–membrane interaction (16Oda S. Noda T. Wijesinghe K.J. Halfmann P. Bornholdt Z.A. Abelson D.M. Armbrust T. Stahelin R.V. Kawaoka Y. Saphire E.O. Crystal structure of Marburg virus VP40 reveals a broad, basic patch for matrix assembly and a requirement of the N-terminal domain for immunosuppression.J. Virol. 2016; 90: 1839-1848Crossref PubMed Scopus (26) Google Scholar, 17Wijesinghe K.J. Stahelin V. Investigation of the lipid binding properties of the Marburg virus matrix protein VP40.J. Virol. 2016; 90: 3074-3085Crossref Scopus (17) Google Scholar, 23Bornholdt Z.A. Noda T. Abelson D.M. Halfmann P. Wood M.R. Kawaoka Y. Saphire E.O. Structural rearrangement of Ebola virus VP40 begets multiple functions in the virus life cycle.Cell. 2013; 154: 763-774Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 24Del Vecchio K. Frick C.T. Gc J.B. Oda S.I. Gerstman B.S. Saphire E.O. Chapagain P.P. Stahelin R.V. A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine.J. Biol. Chem. 2018; 293: 3335-3349Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar); it is also involved in proper protein oligomerization at the lipid membrane (18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 23Bornholdt Z.A. Noda T. Abelson D.M. Halfmann P. Wood M.R. Kawaoka Y. Saphire E.O. Structural rearrangement of Ebola virus VP40 begets multiple functions in the virus life cycle.Cell. 2013; 154: 763-774Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Homo-oligomerization of the filovirus matrix protein is a key and required step for budding of virions (21Adu-Gyamfi E. Johnson K.A. Fraser M.E. Scott J.L. Soni S.P. Jones K.R. Digman M.A. Gratton E. Tessier C.R. Stahelin R.V. Host cell plasma membrane phosphatidylserine regulates the assembly and budding of Ebola virus.J. Virol. 2015; 89: 9440-9453Crossref PubMed Scopus (55) Google Scholar, 22Johnson K.A. Taghon G.J.F. Scott J.L. Stahelin R.V. The Ebola virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2) for extensive oligomerization at the plasma membrane and viral egress.Sci. Rep. 2016; 12: 19125Crossref Scopus (45) Google Scholar, 23Bornholdt Z.A. Noda T. Abelson D.M. Halfmann P. Wood M.R. Kawaoka Y. Saphire E.O. Structural rearrangement of Ebola virus VP40 begets multiple functions in the virus life cycle.Cell. 2013; 154: 763-774Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 25Adu-Gyamfi E. Digman M.A. Gratton E. Stahelin R.V. Single-particle tracking demonstrates that actin coordinates the movement of the Ebola virus matrix protein.Biophys. J. 2012; 103: L41-L53Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 26Nakai K. Okamoto T. Kimura-Someya T. Ishii K. Lim C.K. Tani H. Matsuo E. Abe T. Mori Y. Suzuki T. Miyamura T. Nunberg J.H. Moriishi K. Matsuura Y. Oligomerization of Hepatitis C virus core protein is crucial for interaction with the cytoplasmic domain of E1 envelope protein.J. Virol. 2006; 80: 11265-11273Crossref PubMed Scopus (44) Google Scholar, 27Hilsch M. Goldenbogen B. Sieben C. Höfer C.T. Rabe J.P. Klipp E. Herrmann A. Chiantia S. Influenza A matrix protein M1 multimerizes upon binding to lipid membranes.Biophys. J. 2014; 107: 912-923Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 28Freed E.O. HIV-1 assembly, release and maturation.Nat. Rev. Microbiol. 2015; 13: 484-496Crossref PubMed Scopus (264) Google Scholar). mVP40 and eVP40 are 34% identical in their amino acid sequence but only 16% identical in their CTDs, which gives rise to their different lipid binding selectivity. Differences in their CTDs may also contribute to differences in their oligomerization at the plasma membrane and within the cell. Indeed, mVP40 was previously described as forming large structures in cells (29Timmins J. Schoehn G. Ricard-Blum S. Scianimanico S. Vernet T. Ruigrok R.W.H. Weissenhorn W. Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.J. Mol. Biol. 2003; 326: 493-502Crossref PubMed Scopus (169) Google Scholar, 30Liu Y. Cocka L. Okumura A. Zhang Y.-A. Sunyer J.O. Harty R.N. Conserved motifs within Ebola and Marburg virus VP40 proteins are important for stability, localization, and subsequent budding of virus-like particles.J. Virol. 2010; 84: 2294-2303Crossref PubMed Scopus (42) Google Scholar) and an octamer ring was observed when only the NTD (1–186 aa) was purified (29Timmins J. Schoehn G. Ricard-Blum S. Scianimanico S. Vernet T. Ruigrok R.W.H. Weissenhorn W. Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.J. Mol. Biol. 2003; 326: 493-502Crossref PubMed Scopus (169) Google Scholar). Timmins et al. (29Timmins J. Schoehn G. Ricard-Blum S. Scianimanico S. Vernet T. Ruigrok R.W.H. Weissenhorn W. Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.J. Mol. Biol. 2003; 326: 493-502Crossref PubMed Scopus (169) Google Scholar) hypothesized that the paucity of distinct higher-ordered mVP40 oligomeric structures was a result of the extremely high propensity of mVP40 (1–186 aa) to oligomerize, indicated by the presence of extensive stacked rings (29Timmins J. Schoehn G. Ricard-Blum S. Scianimanico S. Vernet T. Ruigrok R.W.H. Weissenhorn W. Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.J. Mol. Biol. 2003; 326: 493-502Crossref PubMed Scopus (169) Google Scholar). The same investigation successfully captured four distinct eVP40 oligomeric states, suggesting that mVP40 and eVP40 oligomerization may have fundamental differences (29Timmins J. Schoehn G. Ricard-Blum S. Scianimanico S. Vernet T. Ruigrok R.W.H. Weissenhorn W. Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.J. Mol. Biol. 2003; 326: 493-502Crossref PubMed Scopus (169) Google Scholar). Furthermore, the dimeric and hexameric eVP40 crystal structures were resolved in 2013 lending significant insight to the origins of eVP40 lipid binding and oligomerization (23Bornholdt Z.A. Noda T. Abelson D.M. Halfmann P. Wood M.R. Kawaoka Y. Saphire E.O. Structural rearrangement of Ebola virus VP40 begets multiple functions in the virus life cycle.Cell. 2013; 154: 763-774Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The first proposed model of eVP40 oligomerization and arrangement in 2013, VP40 was shown to form hexamers as building blocks of the matrix layer, postulating that electrostatic interactions facilitate the disengagement of the VP40 CTD from the NTD during matrix assembly. This disengagement sets up a conformational change that exposes two key residues within the NTD, Trp95 and Glu160, as part of an oligomeric interface. In 2016, the dimeric structure of mVP40 was resolved (16Oda S. Noda T. Wijesinghe K.J. Halfmann P. Bornholdt Z.A. Abelson D.M. Armbrust T. Stahelin R.V. Kawaoka Y. Saphire E.O. Crystal structure of Marburg virus VP40 reveals a broad, basic patch for matrix assembly and a requirement of the N-terminal domain for immunosuppression.J. Virol. 2016; 90: 1839-1848Crossref PubMed Scopus (26) Google Scholar) revealing a conserved Trp (Trp83) and Asn (Asn148) in mVP40 that align with eVP40-Trp95 and Glu160 (Fig. 1A, NTD panel), respectively. In a previous study, we reported that the Trp83 residue was in a region that exhibited significant shielding during mVP40 membrane association using hydrogen–deuterium exchange-mass spectrometry (HDX-MS) analysis (18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), suggesting it may be important for mVP40 oligomerization following binding to anionic lipids. Furthermore, the previous work demonstrated a reduction of deuterium exchange at the CTD involving residues Leu226 and Ser229 when mVP40 was bound to anionic membranes ((18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), Fig. 1A). Of note, this region, dubbed α-helix 4 (α4 helix), just underlies the lipid-binding surface and is distinct in residue composition and in structure when compared with eVP40. Therefore, we postulated two separate oligomerization interfaces within dimeric mVP40, one involving the CTD α4 helix and a conserved interface within the NTD, as key regulators of mVP40 oligomerization (18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In this study, the initial model of VP40 oligomerization was used. This model involved the formation of VP40 hexamer at the membrane as a building block of the viral matrix assembly. However, a new model of linear arrangement of dimeric VP40 forming a 2D lattice that assembles across the virion membrane has been reported (31Wan W. Clarke M. Norris M.J. Kolesnikova L. Koehler A. Bornholdt Z.A. Becker S. Saphire E.O. Briggs J.A. Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.Elife. 2020; 9e59225Crossref PubMed Scopus (8) Google Scholar). In this model, the linear arrangement of VP40 was shown to be mediated by CTD–CTD interactions (31Wan W. Clarke M. Norris M.J. Kolesnikova L. Koehler A. Bornholdt Z.A. Becker S. Saphire E.O. Briggs J.A. Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.Elife. 2020; 9e59225Crossref PubMed Scopus (8) Google Scholar). To determine the mechanism of lipid-induced mVP40 oligomerization, we assessed different in vitro lipid binding assays with HDX-MS analysis to study the effect of mutations at potential NTD and/or CTD oligomerization interfaces in mVP40 conformational changes upon binding membranes. Then, we conducted cellular studies to rationally investigate how the NTD and CTD oligomerization interfaces coordinate the matrix of mVP40 at the plasma membrane. Findings described here demonstrate that each oligomerization interface mutant displays a significant defect in VLP budding, impairment in overall and proper mVP40 trafficking, and oligomerization at the plasma membrane. In order to better understand the origins of mVP40 oligomers, we constructed the mVP40 hexamer–hexamer interface using the eVP40 hexamer–hexamer interface as the template (Protein Data Bank ID: 4LDD) and performed a 100-ns molecular dynamics simulation. Figure 1B shows the section of the mVP40 filament composed of two hexamers next to each other involving CTD–CTD interactions (Fig. S1A). To test our hypothesis that both the conserved NTD and newly identified residues in the CTD are involved in mVP40 oligomerization, we first generated several mutant constructs. These included the NTD oligomerization interface double mutant W83R/N148A and a CTD oligomerization interface double mutant L226R/S229A. Size exclusion chromatography (SEC) of purified proteins indicated that all proteins formed dimers in solution (Fig. S2). To dissect changes in mVP40 residue solvent accessibility and oligomerization in the absence and presence of membranes, HDX-MS experiments were performed on mVP40 mutants incubated with liposomes containing 45% phosphatidylserine (% molar ratio) as described previously (18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In Figure 1C, the ribbon map of the double mutant W83R/N148A indicates the differences in deuterium incorporation (%D) of the protein in presence of PS-containing liposomes compared with the protein alone. Overall, this double mutant showed little detectable changes in HD exchange pattern in both the NTD (from residue Met1 to Lys47) and CTD (from residue Met263 to Ala284). Similarly, residues Lys96-Gly106 on the helix α1 and residues Gln112-Phe120 on the β4-β5 strands exhibited slightly more rapid HD exchanges. Helix α1 is involved in the dimerization of mVP40, and it had been shown previously that the HD exchange at this region is slower in the presence of anionic lipid-containing liposomes (18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The HDX-MS profile of W83R/N148A also showed an increase of HD exchange at the β6 strand (residues Glu140-Gln146) as well as in the region Met261 to Gln276, which is in basic loop 2 and the β10 strand. Oda et al. (16Oda S. Noda T. Wijesinghe K.J. Halfmann P. Bornholdt Z.A. Abelson D.M. Armbrust T. Stahelin R.V. Kawaoka Y. Saphire E.O. Crystal structure of Marburg virus VP40 reveals a broad, basic patch for matrix assembly and a requirement of the N-terminal domain for immunosuppression.J. Virol. 2016; 90: 1839-1848Crossref PubMed Scopus (26) Google Scholar) showed that residues in this region are involved in the efficient binding of mVP40 to PS-containing liposomes. All together, these results suggest that the residues Trp83 and Asn148 are involved in the formation of oligomers that shields these specific regions from exposure to the aqueous environment resulting in slow deuterium incorporation/exchange rates upon binding to PS-containing lipid vesicles. Furthermore, the double mutant W83R/N148A exhibited an intermediate change in deuteration level compared with wildtype mVP40 (WT-mVP40) in the presence or absence of zwitterionic phospholipid (Fig. S1B adapted from (18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar)). Next, we analyzed the solvent accessibility of the CTD double mutant L226R/S229A upon binding to PS-containing lipid vesicles. Similar to W83R/N148A, L226R/S229A exhibited an overall increase of the HD exchange profile compared with WT-mVP40 (Fig. S1B), except in the region including residues Ile88-Asn91. Furthermore, no changes of the deuteration level of the β6 strand (residues Glu140-Phe145) were observed for L226R/S229A compared with WT-mVP40, which showed very slow HD exchange in the presence of PS-containing vesicles within the same region (18Wijesinghe K.J. Urata S. Bhattarai N. Kooijman E.E. Gerstman B.S. Chapagain P.P. Li S. Stahelin R.V. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.J. Biol. Chem. 2017; 292: 6108-6122Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). As mentioned above, L226R/S229A-mVP40 showed a faster HD exchange profile than WT-mVP40, including the following regions: i) in the NTD from residue Tyr13 to Tyr44 (which contains the β1 strand), residues Glu73-Gly87 (unstructured loop between β2-β3 strands and the N terminus of β3 strand), Phe113-Phe120 (β4-β

Referência(s)