Functional mapping of the nucleoprotein of Ebola virus

Abstract

At 739 amino acids, the nucleoprotein (NP) of Ebola virus is the largest nucleoprotein of the nonsegmented negative-stranded RNA viruses, and like the NPs of other viruses, it plays a central role in virus replication. Huang et al. (Y. Huang, L. Xu, Y. Sun, and G. J. Nabel, Mol. Cell 10:307-316, 2002) previously demonstrated that NP, together with the minor matrix protein VP24 and polymerase cofactor VP35, is necessary and sufficient for the formation of nucleocapsid-like structures that are morphologically indistinguishable from those seen in Ebola virus-infected cells. They further showed that NP is O glycosylated and sialylated and that these modifications are important for interaction between NP and VP35. However, little is known about the structure-function relationship of Ebola virus NP. Here, we examined the glycosylation of Ebola virus NP and further investigated its properties by generating deletion mutants to define the region(s) involved in NP-NP interaction (self-assembly), in the formation of nucleocapsid-like structures, and in the replication of the viral genome. We were unable to identify the types of glycosylation and sialylation, although we did confirm that Ebola virus NP was glycosylated. We also determined that the region from amino acids 1 to 450 is important for NP-NP interaction (self-assembly). We further demonstrated that these amino-terminal 450 residues and the following 150 residues are required for the formation of nucleocapsid-like structures and for viral genome replication. These data advance our understanding of the functional region(s) of Ebola virus NP, which in turn should improve our knowledge of the Ebola virus life cycle and its extreme pathogenicity.

FIG. 1.

Characterization of Ebola virus NP glycosylation. (A) Detection of [³H]glucosamine-labeled Ebola virus NP. IP, immunoprecipitation. (B) Purification of Ebola virus NP, transcription factor Sp1, and a soluble form of Ebola virus GP. Purified proteins were subjected to SDS-PAGE and stained with Coomassie brilliant blue. The asterisk indicates Sp1; the lower band is probably a protein nonspecifically bound to and eluted from the column. (C) No nucleocytoplasmic O glycosylation was detected on Ebola virus NP with an anti-GlcNAc antibody. (D) No mucin-type O glycosylation or sialylation was detected on Ebola virus NP by using lectins: peanut agglutinin (PNA), Sambucus nigra agglutinin (SNA), and Maackia amurensis agglutinin (MAA).

FIG. 2.

Ebola virus NP interacts with itself. Plasmids encoding His- or FLAG-tagged Ebola virus NP were transfected separately (NP-H or NP-F) or together (NP-H+NP-F) into 293T cells. Proteins were detected with an anti-His or an anti-FLAG antibody either before (Total cell lysate) or after immunoprecipitation with the indicated antibody. IP denotes immunoprecipitation.

FIG. 3.

Interaction of wild-type NP with its mutants. (A) Schematic diagram of NP deletion mutants. A series of NP deletion mutants was constructed by sequential deletion of 150 N-terminal amino acids. A FLAG tag was added to the C-terminal end of the wild type and the mutants. (B) Expression of Ebola virus NP mutants. 293T cells were transfected with a plasmid encoding either wild-type FLAG-tagged NP or a FLAG-tagged mutant NP. Cell lysates were subjected to SDS-PAGE, followed by Western blotting using an anti-FLAG antibody. (C) Interaction of wild-type His-tagged NP with FLAG-tagged mutant NPs. A plasmid encoding wild-type His-tagged NP was cotransfected with a plasmid encoding either wild-type FLAG-tagged NP or FLAG-tagged mutant NP into 293T cells. Cell lysates were subjected to immunoprecipitation with either anti-FLAG or anti-His antibody. The immunoprecipitated proteins were analyzed by Western blotting using anti-FLAG and anti-His antibodies. IP denotes immunoprecipitation.

FIG. 4.

The N-terminal half of Ebola virus NP is important for NP-NP interaction. (A) Schematic diagram of a deletion mutant lacking amino acid residues at positions 451 to 739 (Δ451-739F). (B) Expression of a mutant NPΔ451-739F. 293T cells were transfected with a plasmid encoding either wild-type FLAG-tagged NP or FLAG-tagged NPΔ451-739. Cell lysates were subjected to SDS-PAGE, followed by Western blotting using an anti-FLAG antibody. (C) Interaction of wild-type His-tagged NP with FLAG-tagged NPΔ451-739. A plasmid encoding wild-type His-tagged NP was cotransfected with a plasmid encoding either wild-type FLAG-tagged NP or FLAG-tagged NPΔ451-739 into 293T cells. Cell lysates were subjected to immunoprecipitation with either an anti-FLAG or an anti-His antibody. Immunoprecipitated proteins were analyzed by Western blotting using anti-FLAG and anti-His antibodies. The band marked by an asterisk in panels B and C likely represents a dimeric form of the mutant FLAG-tagged NPΔ451-739. IP denotes immunoprecipitation.

FIG.5.

Electron microscopy of NP deletion mutants. (A) Electron micrographs showing Ebola virus-infected cells containing nucleocapsids; cells expressing NP, VP35, and VP24 and thus possessing nucleocapsid-like structures; and cells expressing NP alone, resulting in the formation of helical-ring-like structures. Bars, 100 nm. (B) Schematic diagram of NP tube and nucleocapsid-like structures. (C and D) Formation of NP tube and nucleocapsid-like structures in NP mutant-expressing cells. Wild-type or individual mutants were expressed in the absence (C) or presence (D) of VP35 and VP24 in 293T cells. WT, wild type. Bars, 200 nm.

FIG.5.

Electron microscopy of NP deletion mutants. (A) Electron micrographs showing Ebola virus-infected cells containing nucleocapsids; cells expressing NP, VP35, and VP24 and thus possessing nucleocapsid-like structures; and cells expressing NP alone, resulting in the formation of helical-ring-like structures. Bars, 100 nm. (B) Schematic diagram of NP tube and nucleocapsid-like structures. (C and D) Formation of NP tube and nucleocapsid-like structures in NP mutant-expressing cells. Wild-type or individual mutants were expressed in the absence (C) or presence (D) of VP35 and VP24 in 293T cells. WT, wild type. Bars, 200 nm.

FIG. 6.

The region of NP important for GFP expression from the Ebola virus minigenome. The wild type or individual mutants were expressed, together with VP35, VP30, L, the Ebola virus minigenome possessing the GFP gene, and the T7 polymerase. GFP expression was detected by Western blotting. Ab and mAb denote antibody and monoclonal antibody, respectively. The asterisk indicates expression of NPΔ451-739, which was barely detectable with this polyclonal antibody.

FIG. 7.

Dominant-negative effect of NP mutants on GFP expression from the Ebola virus minigenome. Various amounts of NP mutants (NPΔ451-600 or NPΔ451-739) were expressed with a constant amount of wild-type NP (WT), along with VP35, VP30, L, the Ebola virus minigenome possessing the GFP gene, and the T7 polymerase. GFP expression was detected by Western blotting.