Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression

Abstract

Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. IMPORTANCEMarburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. Marburg virus (MARV) and the related Ebola virus (EBOV) both belong to the Filoviridae family. These are enveloped viruses with a nonsegmented single-stranded and negative-sense RNA genome. Genomes of Marburg viruses differ from those of ebolaviruses by more than 50% at the nucleotide level (1). MARV causes severe and rapidly progressing hemorrhagic fever in humans and nonhuman primates, with lethality ranging from 25% to over 90% depending on the geographic location and viral strain (2). The 19-kb MARV genome encodes seven structural proteins. Each of these proteins is of critical importance, and most are known to perform multiple functions during the viral life cycle.VP40 is the Marburg virus matrix protein, which builds the protein shell underneath the viral envelope and confers the hallmark filamentous morphology to the Marburg virion. VP40 alone is able to induce the assembly and budding of filamentous viruslike particles (VLPs), which resemble authentic virions (3, 4). The intracellular distribution of filovirus VP40 varies during the progression of the viral life cycle (5, 6), as it orchestrates the distribution of the other viral components and viral assembly (6, 7). The majority of it, however, traffics to and associates with the cellular membrane (5,7,8). Within the virion, VP40 interacts with both the lipid envelope and the core nucleocapsid complex, which contains the NP, VP35, L, VP30, and VP24 proteins (9). VP40 also interacts with the GP viral surface protein. Indeed, coexpression of VP40 and GP alters the distributi...

Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40

“…The cationic amino acid side chains that project away from the CTD surface provide ideal sites to form salt bridges with the anionic phospholipid head groups and mediate membrane interactions. Our data support the idea that mVP40 interactions with the membrane rely heavily on electrostatic effects, which is further supported in the structural analysis, where single mutation of Lys210 to Glu abrogated VLP formation (26). Moreover, we have demonstrated that the K210E construct binding to vesicles containing PS was ϳ30% reduced while binding of double and triple mutations of the mVP40 cationic patch was nearly fully abrogated (26).…”

“…mVP40 binding to membranes is dependent upon the anionic charge density of the plasma membrane, which when neutralized ablates mVP40 localization. The binding and cellular localization of mVP40 are consistent with recent structural analysis demonstrating that mVP40 has a flat and extensive CTD basic surface (26).…”

“…From the recent crystal structure of the mVP40 dimer (26), the CTD harbors the purported lipid binding surface or the basic patch of the protein. The mVP40 dimer is flatter and contains a basic patch larger than that of eVP40.…”

“…eVP40 harbors an N-terminal domain (NTD) involved in oligomerization (16)(17)(18)(19) and a C-terminal domain (CTD) reported to mediate membrane binding (20)(21)(22)(23)(24)(25). Recent structural analyses of eVP40 (21) and mVP40 (26) have revealed an NTD interface that mediates dimerization, which is required for VP40 plasma membrane localization and budding. The NTDs have 42% sequence identity and similar folds, while the CTD is only 15% identical in sequence and somewhat different in structure (26).…”

“…Recent structural analyses of eVP40 (21) and mVP40 (26) have revealed an NTD interface that mediates dimerization, which is required for VP40 plasma membrane localization and budding. The NTDs have 42% sequence identity and similar folds, while the CTD is only 15% identical in sequence and somewhat different in structure (26). This suggests that eVP40 and mVP40 may have different membrane binding properties mediated by CTD interactions with the plasma membrane interface.…”