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Crystal structure of the Marburg virus GP2 core domain in its postfusion conformation.

ABSTRACT: Marburg virus (MARV) and Ebola virus (EBOV) are members of the family Filoviridae ("filoviruses") and cause severe hemorrhagic fever with human case fatality rates of up to 90%. Filovirus infection requires fusion of the host cell and virus membranes, a process that is mediated by the envelope glycoprotein (GP). GP contains two subunits, the surface subunit (GP1), which is responsible for cell attachment, and the transmembrane subunit (GP2), which catalyzes membrane fusion. The GP2 ectodomain contains two heptad repeat regions, N-terminal and C-terminal (NHR and CHR, respectively), that adopt a six-helix bundle during the fusion process. The refolding of this six-helix bundle provides the thermodynamic driving force to overcome barriers associated with membrane fusion. Here we report the crystal structure of the MARV GP2 core domain in its postfusion (six-helix bundle) conformation at 1.9 Å resolution. The MARV GP2 core domain backbone conformation is virtually identical to that of EBOV GP2 (reported previously), and consists of a central NHR core trimeric coiled coil packed against peripheral CHR ?-helices and an intervening loop and helix-turn-helix segments. We previously reported that the stability of the MARV GP2 postfusion structure is highly pH-dependent, with increasing stability at lower pH [Harrison, J. S., Koellhoffer, J. K., Chandran, K., and Lai, J. R. (2012) Biochemistry51, 2515-2525]. We hypothesized that this pH-dependent stability provides a mechanism for conformational control such that the postfusion six-helix bundle is promoted in the environments of appropriately mature endosomes. In this report, a structural rationale for this pH-dependent stability is described and involves a high-density array of core and surface acidic side chains at the midsection of the structure, termed the "anion stripe". In addition, many surface-exposed salt bridges likely contribute to the stabilization of the postfusion structure at low pH. These results provide structural insights into the mechanism of MARV GP2-mediated membrane fusion.

SUBMITTER: Koellhoffer JF

PROVIDER: S-EPMC3464016 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Crystal structure of the Marburg virus GP2 core domain in its postfusion conformation.

Koellhoffer Jayne F JF Malashkevich Vladimir N VN Harrison Joseph S JS Toro Rafael R Bhosle Rahul C RC Chandran Kartik K Almo Steven C SC Lai Jonathan R JR

Biochemistry 20120919 39

Marburg virus (MARV) and Ebola virus (EBOV) are members of the family Filoviridae ("filoviruses") and cause severe hemorrhagic fever with human case fatality rates of up to 90%. Filovirus infection requires fusion of the host cell and virus membranes, a process that is mediated by the envelope glycoprotein (GP). GP contains two subunits, the surface subunit (GP1), which is responsible for cell attachment, and the transmembrane subunit (GP2), which catalyzes membrane fusion. The GP2 ectodomain co ...[more]

PMID: 22935026

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Project description:Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs.IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and provide more details for therapeutic intervention.

Project description:Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV requires fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein, GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔG(unf,H(2)O), of 33.4 ± 2.5 kcal/mol and melting temperature, T(m), of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH, with higher stability under lower-pH conditions (T(m) values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. On the basis of these results, we hypothesize that the pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism for controlling conformational preferences such that the six-helix bundle "postfusion" state is preferred under conditions of appropriately matured endosomes.

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Crystal structure of the Marburg virus GP2 core domain in its postfusion conformation.

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Crystal structure of the Marburg virus GP2 core domain in its postfusion conformation.

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