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Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

ABSTRACT: 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.

SUBMITTER: Zhu T

PROVIDER: S-EPMC5571254 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

Zhu Tengfei T Song Hao H Peng Ruchao R Shi Yi Y Qi Jianxun J Gao George F GF

Journal of virology 20170824 18

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 ...[more]

PMID: 28659479

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Project description:Marburg virus (MARV) encodes a nucleoprotein (NP) to encapsidate its genome by oligomerization and form a ribonucleoprotein complex (RNP). According to previous investigation on nonsegmented negative-sense RNA viruses (nsNSV), the newly synthesized NPs must be prevented from indiscriminately binding to noncognate RNAs. During the viral RNA synthesis process, the RNPs undergo a transition from an RNA-bound form to a template-free form, to open access for the interaction between the viral polymerase and the RNA template. In filoviruses, this transition is regulated by VP35 peptide and other viral components. To further understand the dynamic process of filovirus RNP formation, we report here the structure of MARV NPcore, both in the apo form and in the VP35 peptide-chaperoned form. These structures reveal a typical bilobed structure, with a positive-charged RNA binding groove between two lobes. In the apo form, the MARV NP exists in an interesting hexameric state formed by the hydrophobic interaction within the long helix of the NPcore C-terminal region, which shows high structural flexibility among filoviruses and may imply critical function during RNP formation. Moreover, the VP35 peptide-chaperoned NPcore remains in a monomeric state and completely loses its affinity for single-stranded RNA (ssRNA). The structural comparison reveals that the RNA binding groove undergoes a transition from closed state to open state, chaperoned by VP35 peptide, thus preventing the interaction for viral RNA. Our investigation provides considerable structural insight into the filovirus RNP working mechanism and may support the development of antiviral therapies targeting the RNP formation of filovirus.IMPORTANCE Marburg virus is one of the most dangerous viruses, with high morbidity and mortality. A recent outbreak in Angola in 2005 caused the deaths of 272 persons. NP is one of the most essential proteins, as it encapsidates and protects the whole virus genome simultaneously with self-assembly oligomerization. Here we report the structures of MARV NPcore in two different forms. In the MARV NP apo form, we identify an interesting hexamer formed by hydrophobic interaction within a long helix, which is highly conserved and flexible among filoviruses and may indicate its critical function during the virus RNP formation. Moreover, the structural comparison with the NP-VP35 peptide complex reveals a structural transition chaperoned by VP35, in which the RNA binding groove undergoes a transition from closed state to open state. Finally, we discussed the high conservation and critical role of the VP35 binding pocket and its potential use for therapeutic development.

Project description:The filoviruses are etiological agents of life-threatening hemorrhagic fever with high mortality rate and risk of potential outbreak. Among members of this family, the Ebola (EBOV), Sudan (SUDV), and Marburg (MARV) viruses are considered the most pathogenic for humans. The ebolavirus nucleoprotein (NP) is the most abundant protein in infected cells and is essential for viral transcription and replication; thus, it represents an attractive target for therapeutic intervention. Here, we present the structure of SUDV NP in complex with the amino-terminal portion of the phosphoprotein VP35 at 2.3 Å. This structure captures VP35 chaperoning SUDV NP in a monomeric and RNA-free state. This transient state has been proposed to be key to maintaining a pool of monomeric and RNA-free NPs prior to NP-NP polymerization and encapsidation of the viral RNA genome. This structure also reveals a newly visualized interaction between NP and VP35, a well-defined beta sheet that is not present in previous structures. Affinity binding assays demonstrate that this beta sheet is essential for maintaining the high-affinity interaction between VP35 and a hydrophobic pocket on SUDV NP, and electron microscopy indicates the importance of this binding interaction to the oligomeric state and assembly of NP in human cells. Complementary structure-directed mutagenesis identifies critical residues conserved across the filovirus family that could be targeted by broadly effective antivirals.IMPORTANCE Outbreaks of the filoviruses can be unpredictable in timing, location, and identity of the causative virus, with each of Ebola virus, Sudan virus, Bundibugyo virus, and Marburg virus reemerging in the last several years to cause human disease with 30 to 90% lethality. The 2014-2016 outbreak in particular, with nearly 30,000 patients, highlighted the ability of these viruses to emerge unexpectedly and spread rapidly. Two ebolavirus outbreaks have emerged this year, yet we still lack FDA-approved drugs with pan-filovirus activity to treat existing and emergent ebolaviruses. For all filoviruses, the interaction between the nucleoprotein and the phosphoprotein is essential for the virus life cycle and is a potential target for therapeutic intervention. In this report, we describe the crystal structure of the SUDV nucleoprotein with the interacting domain of the viral phosphoprotein, and we identify residues critical for high-affinity interaction and for control of the oligomeric state of the nucleoprotein. Structural comparison of this heterodimer with other members of the filovirus family allowed us to find conserved and essential atomic features that will facilitate understanding of the virus life cycle and the rational design of antivirals.

Project description:The Ebola virus and its close relative, the Marburg virus, both belong to the family Filoviridae and are highly hazardous and contagious viruses. With a mortality rate ranging from 23% to 90%, depending on the specific outbreak, the development of effective antiviral interventions is crucial for reducing fatalities and mitigating the impact of Marburg virus outbreaks. In this investigation, a virtual screening approach was employed to evaluate 2042 natural compounds for their potential interactions with the VP35 protein of the Marburg virus. Average and worst binding energies were calculated for all 20 poses, and compounds that exhibited binding energies <-6 kcal/mol in both criteria were selected for further analysis. Based on binding energies, only six compounds (Estradiol benzoate, INVEGA (paliperidone), Isosilybin, Protopanaxadiol, Permethrin, and Bufalin) were selected for subsequent investigations, focusing on interaction analysis. Among these selected compounds, Estradiol benzoate, INVEGA (paliperidone), and Isosilybin showed strong hydrogen bonds, while the others did not. In this study, the compounds Myricetin, Isosilybin, and Estradiol benzoate were subjected to a molecular dynamics (MD) simulation and free binding energy calculation using MM/GBSA analysis. The reference component Myricetin served as a control. Estradiol benzoate exhibited the most stable and consistent root-mean-square deviation (RMSD) values, whereas Isosilybin showed significant fluctuations in RMSD. The compound Estradiol benzoate exhibited the lowest ΔG binding free energy (-22.89 kcal/mol), surpassing the control compound's binding energy (-9.29 kcal/mol). Overall, this investigation suggested that Estradiol benzoate possesses favorable binding free energies, indicating a potential inhibitory mechanism against the VP35 protein of the Marburg virus. The study proposes that these natural compounds could serve as a therapeutic option for preventing Marburg virus infection. However, experimental validation is required to further corroborate these findings.

Project description: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 α-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.

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Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

Publications

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

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