The Ebola virus ribonucleoprotein complex: A novel VP30–L interaction identified

National Laboratory for Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg R3E 3R2, Canada.
Virus Research (Impact Factor: 2.32). 12/2008; 140(1-2):8-14. DOI: 10.1016/j.virusres.2008.10.017
Source: PubMed


The ribonucleoprotein (RNP) complex of Ebola virus (EBOV) is known to be a multiprotein/RNA structure, however, knowledge is rather limited regarding the actual protein-protein interactions involved in its formation. Here we show that singularly expressed VP35 and VP30 are present throughout the cytoplasm, while NP forms prominent cytoplasmic inclusions and L forms smaller perinuclear inclusions. We could demonstrate the existence of NP-VP35, NP-VP30 and VP35-L interactions, similar to those described for Marburg virus (MARV) based on the redistribution of protein partners into NP and L inclusion bodies. Significantly, a novel VP30-L interaction was also identified and found to form as part of an NP-VP30-L bridge structure, similar to that formed by VP35. The identification of these interactions allows a preliminary model of the EBOV RNP complex structure to be proposed, and may provide insight into filovirus transcriptional regulation.

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    • "The polymerase cofactor VP35 is the functional equivalent of the phosphoprotein P of other NNS RNA viruses (Mühlberger et al., 1999). It interacts with L and NP leading to the formation of trimeric complexes in which VP35 serves as a bridge between NP and L (Becker et al., 1998; Boehmann et al., 2005; Groseth et al., 2009). In addition, VP35 forms homo-oligomers mediated by an amino-terminally located coiled-coil motif (Reid et al., 2005; Zinzula et al., 2009). "
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    ABSTRACT: The Ebola virus (EBOV) RNA-dependent RNA polymerase (RdRp) complex consists of the catalytic subunit of the polymerase, L, and its cofactor VP35. Using immunofluorescence analysis and coimmunoprecipitation assays, we mapped the VP35 binding site on L. A core binding domain spanning amino acids 280-370 of L was sufficient to mediate weak interaction with VP35, while the entire N-terminus up to amino acid 380 was required for strong VP35-L binding. Interestingly, the VP35 binding site overlaps with an N-terminal L homo-oligomerization domain in a non-competitive manner. N-terminal L deletion mutants containing the VP35 binding site were able to efficiently block EBOV replication and transcription in a minigenome system suggesting the VP35 binding site on L as a potential target for the development of antivirals.
    Virology 04/2013; 441(2). DOI:10.1016/j.virol.2013.03.013 · 3.32 Impact Factor
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    • "We propose that VP30 lies in the interior of the nucleocapsid and is not part of the bridge on the periphery of the nucleocapsid. This localization is consistent with previous work showing that VP30 is a component of the nucleocapsid, and associates with NP, but is non-essential for nucleocapsid formation [14], [30], [31]. Our model, in which the inner layer at 22.3 nm diameter is RNA-NP, and the outer bridge centered at 37 nm is composed of VP24-VP35 heterodimers, with VP30 bound to NP, is thus consistent with these previous observations [14], [28], [29], and suggests that the outer VP24-VP35 heterodimer bridge functions in the stabilization and/or protection of the nucleocapsid. "
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    ABSTRACT: Filoviruses, including Ebola virus, are unusual in being filamentous animal viruses. Structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level 4 biological containment. The present study provides unique insights into the structure of this deadly pathogen. We have investigated the structure of Ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. Here we report the three-dimensional structure and architecture of Ebola virus and establish that multiple copies of the RNA genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. We show that the helical Ebola virus inner nucleocapsid containing RNA and nucleoprotein is stabilized by an outer layer of VP24-VP35 bridges. Elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. The matrix protein VP40 forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular. The results of this study demonstrate a modular organization in Ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope.
    PLoS ONE 01/2012; 7(1):e29608. DOI:10.1371/journal.pone.0029608 · 3.23 Impact Factor
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    • "In addition to NP, the RNP complex also contains the viral RNA genome, the polymerase (L), the polymerase cofactor (VP35) and the transcriptional activator (VP30). NP has been shown to directly interact with VP35 and VP30, both of which interact with L (Becker et al., 1998; Groseth et al., 2009). "
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    ABSTRACT: The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40-RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.
    Journal of General Virology 09/2011; 92(Pt 12):2900-5. DOI:10.1099/vir.0.036863-0 · 3.18 Impact Factor
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