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

https://doi.org/10.1016/j.virusres.2008.10.017Get rights and content

Abstract

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.

Introduction

The most recent classification divides Filoviridae into two genera, Marburgvirus and Ebolavirus. While the genus Marburgvirus consists of a single species, Lake Victoria marburgvirus (MARV), the genus Ebolavirus (EBOV) is subdivided into four species, Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Cote d’Ivoire ebolavirus (CIEBOV), and Reston ebolavirus (REBOV) (Feldmann et al., 2004). A putative fifth species has also been postulated, which is the cause of a recent outbreak in Uganda (Towner et al., 2008). Apart from the obvious phylogenetic division between MARV and EBOV based on nucleotide sequence, they are further distinguished by their general lack of antigenic cross-reactivity and differences in their genome organization. However, most viral processes and the functions of the viral proteins are presumed to be identical between MARV and EBOV (Sanchez et al., 2007).

Of the seven structural proteins, four of these, together with the viral RNA, make up the ribonucleoprotein (RNP) complex (Elliott et al., 1985, Mühlberger et al., 1998, Mühlberger et al., 1999, Mühlberger, 2004, Sanchez et al., 2007). Within this RNP complex the nucleoprotein (NP) functions in RNA encapsidation, virion protein (VP) 35 acts as an RNA-dependent RNA polymerase cofactor, VP30 is important structurally as a minor nucleoprotein, as well as acting as an EBOV-specific transcriptional activator, and L functions as the RNA-dependent RNA polymerase (Elliott et al., 1985, Mühlberger et al., 1998, Mühlberger et al., 1999, Mühlberger, 2004, Sanchez et al., 2007). These four proteins also represent the minimal necessary factors for the transcription and replication of the EBOV genome, although VP30 has been shown to be dispensable for replication alone (Mühlberger et al., 1999). Interestingly, despite the presence of MARV VP30 in the RNP complex, it is not required for either the transcription or replication of MARV minigenomes (Mühlberger et al., 1998). Although a mechanistic basis for this difference has not yet been established, current evidence suggests a role for VP30 in overcoming a hairpin structure overlapping the NP transcriptional start site in EBOV (Weik et al., 2002). However, it has been recently reported that VP30 is necessary for the rescue of MARV using an infectious clone system, independent of residues important for its transcriptional activator function in EBOV, suggesting that VP30 serves additional functions critical for transcription when in the context of a full-length genome (Enterlein et al., 2006).

It has been previously shown that within the RNP complex of MARV both NP and L interact with VP35, but do not directly interact with each other (Becker et al., 1998). Thus VP35 serves as a bridging molecule, which is believed to recruit L to the encapsidated RNA (Becker et al., 1998). Based on more recent data, it also appears that oligomerization of MARV VP35 is necessary for the interaction with L, but not for interaction with NP (Möller et al., 2005), suggesting that interaction of NP and VP35 also occurs separately from interaction between VP35 and L. In addition, it was shown that MARV VP30 interacts directly with NP, a process that is likely essential for its function as a minor nucleoprotein (Becker et al., 1998). For EBOV no such systematic attempt has been made to address the interactions existing within the RNP complex. However, it is apparent from various studies that interaction occurs between NP and VP30 (Modrof et al., 2002), as well as NP and VP35 (Huang et al., 2002, Watanabe et al., 2006). Information regarding the interactions of L within the RNP complex has not yet been reported, likely due to the absence of antibodies available to detect the polymerase. Therefore, it was the purpose of this study to develop the necessary resources to facilitate detection of each of the EBOV RNP components in order to identify the protein–protein interactions involved in formation of the RNP complex of EBOV, with a particular focus on interaction partners for the polymerase.

Section snippets

Cells

Vero E6 (African green monkey kidney), 293T (human embryonic kidney) and Ad-293 (human embryonic kidney; Stratagene) cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin and 100 (g/mL streptomycin and grown at 37 °C and 5% CO2. Escherichia coli (E. coli) of the XL-1 Blue strain were used for all routine cloning procedures, while BL-21 E. coli (GE Healthcare), deficient in the OmpT and Lon proteases, were used for the expression of

Production and characterization of antibodies

The availability of immunological reagents for filoviruses was, and continues to be, limited, particularly for targets other than the glycoprotein (GP). In order to address this need, and allow detection of the RNP proteins of REBOV, antisera were raised against bacterially expressed GST fusion proteins, containing the VP35 ORF, the VP30 ORF or peptide sequences from NP or L (Fig. 1A). Since the NP and L proteins were too large to allow expression in their entirety as a GST fusion, peptides

Acknowledgements

The authors are grateful to Victoria Wahl-Jensen, for assistance with various immunofluorescence protocols, and to Hideki Ebihara for valuable discussion. This work was supported by a grant of the Canadian Institutes of Health Research (MOP–43921, awarded to H.F.), and scholarships of the Natural Sciences and Engineering Research Council of Canada (A.G.), the German Academic Exchange Service (M.S.) and the German Chemical Industry Association (T.H.), as well as by funding from the Public Health

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