Ebola virus transcription activator VP30 is a zinc-binding protein

Abstract

Ebola virus VP30 is an essential activator of viral transcription. In viral particles, VP30 is closely associated with the nucleocapsid complex. A conspicuous structural feature of VP30 is an unconventional zinc-binding Cys(3)-His motif comprising amino acids 68 to 95. By using a colorimetric zinc-binding assay we found that the VP30-specific Cys(3)-His motif stoichiometrically binds zinc ions in a one-to-one relationship. Substitution of the conserved cysteines and the histidine within the motif led to a complete loss of the capacity for zinc binding. Functional analyses revealed that none of the tested mutations of the proposed zinc-coordinating residues influenced binding of VP30 to nucleocapsid-like particles but, concerning its role in activating viral transcription, all resulted in a protein that was inactive.

FIG. 1.

Conserved Cys₃-His motifs within viral proteins and expression of GST-VP30_68-95 fusion proteins. (A) Alignment of a Cys₃-His zinc-binding motif consensus sequence (29) and VP30 sequences of different filoviruses as well as the M2-1 protein sequence of hRSV. Predicted zinc-coordinating residues are printed in bold. MBGV, Marburg virus. (B) Diagram of EBOV VP30-specific peptides expressed as GST fusion proteins. GST (in boxes) represents the amino-acid sequence of GST. The C-terminally fused VP30-specific amino acids are listed. Mutated amino acids are underlined. (C) Bacterial expression and purification of GST fusion proteins. Purified proteins were analyzed by SDS-PAGE in 12% polyacrylamide gels and visualized by staining with Coomassie blue.

FIG. 2.

Amino acids 68 to 95 of VP30 bind zinc in vitro. (A) Binding of zinc ions by GST and GST-Z as determined by measuring optical density at 490 nm in the presence of 0.1 mM PAR following the Z→E or E→Z incubation series. For Z→E incubation, the purified proteins were first incubated with 0.1 mM zinc acetate and subsequently with 1 mM EDTA; for E→Z incubation, purified proteins were first incubated with 1 mM EDTA and afterwards with 0.1 mM zinc acetate. (B) Binding of zinc ions by GST, GST-Z, GST-Z_II, GST-Z_III, and GST-Z_IV after treatment with 0.1 mM zinc acetate. Standard deviations are indicated by bars. n, number of independent experiments.

FIG. 3.

Mutations within the zinc-binding motif do not influence binding to NP-induced inclusions. (A) Diagram of the mutations inserted in the Cys₃-His motif of the full-length VP30. Mutated amino acids are underlined. (B) HeLa cells grown on glass coverslips were infected with MVA-T7 and cotransfected with 200 ng of the DNA plasmids encoding the wild-type VP30 or the VP30 substitution mutants together with 1 μg of pT-NP_EBO (encoding EBOV NP). At 12 h postinfection, cells were fixed, permeabilized using 0.2% Triton X-100, and subjected to immunofluorescence analysis using a monoclonal anti-NP immunoglobulin G (IgG) antibody (1:20) and a monoclonal anti-VP30 IgM antibody (1:10). As secondary antibodies, a μ chain-specific fluorescein isothiocyanate-conjugated F(ab′)₂ fragment of goat anti-mouse IgM (1:100; Dianova) and a rhodamine-conjugated goat anti-mouse IgG (1:100; Dianova) were used. Panel A, NP and VP30 expressed alone; panel B, coexpression of VP30 and NP; panels C to E, coexpression of VP30 mutants with NP; panel F, MVA-T7-infected cells.

FIG. 4.

Transcription activation mediated by VP30 requires an intact zinc-binding motif. Approximately 5 × 10⁵ BSR T7/5 cells were transfected with DNA plasmids encoding EBOV nucleocapsid proteins NP, VP35, L, and either the VP30 or the VP30 mutant, together with a DNA plasmid encoding the EBOV-specific artificial minigenome 3E-5E. At 2 days posttransfection, cells were lysed and CAT activity was determined. Lane 1, control without VP30; lane 2, wild-type VP30; lanes 3 to 5, VP30 substitution mutants (the names of the respective mutants are given at the top of the panel).