Ebola Virus VP30 Is an RNA Binding Protein

Abstract

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn Ebola viruses (EBOV) are nonsegmented, negative-stranded RNA viruses, which together with Marburg virus, constitute the family Filoviridae (18). Filoviruses cause severe and lethal hemorrhagic fevers in humans and nonhuman primates and, as such, are classified as biosafety level 4 (BSL-4) agents (11). Of the eight proteins encoded by the EBOV genome, the L protein, VP35, the N protein (NP), and VP30 are proposed to form a ribonucleocapsid complex, along with the genomic RNA (10, 28). The L protein bears the enzymatic activities required for transcription and replication, whereas VP30 and VP35 are implicated in regulation of transcription and replication (28,41). Although the NP is necessary for transcription, its role is still unknown (28). Phosphorylation of VP30 positively regulates its binding to NP and negatively regulates its transcriptional activity (26). In addition, enhancement of transcription by VP30 requires a putative RNA secondary structure located within nucleotides (nt) 54 to 80 of the leader region (44). Deletion of the predicted RNA secondary structure permits VP30-independent transcription of viral messengers (44). These reported activities of VP30 suggest the possibility of a direct interaction of VP30 with EBOV RNA in its role in transcription. In addition, the N terminus of VP30 contains a Zn 2ϩ binding Cys 3 -His motif (25) and is rich in basic amino acids (34), which can interact with nucleic acid, and provided impetus for our studies here. By analogy, a homologue of VP30, the M2-1 protein of human respiratory syncytial virus (hRSV), has a Cys 3 -His motif that binds hRSV leader RNA with an apparent dissociation constant of 90 nM (8,14). Mutation of three cysteine residues of the Cys 3 -His motif led to a significant reduction of hRSV recovery (39).Recent publications with the minigenome system for EBOV suggest at least two possible mechanisms that VP30 may use in its transcriptional regulatory role. One possible mechanism could be that VP30 interacts with one or more of the other nucleocapsid proteins, polymerase, NP or VP35, and promotes increased stability of the transcriptional complex. An increase in the number of active transcription initiation complexes or the increased stability of the transcriptional...

How disordered is my protein and what is its disorder for? A guide through the “dark side” of the protein universe

“…Due to its high sensitivity to local sequence peculiarities, PONDR Ò VLXT was discovered to be able to identify disorder-based interaction sites 86 (for examples see refs. 164,165). HCA is similarly instrumental for the identification of regions undergoing induced folding, as buried hydrophobic residues at the protein-partner interface are often the major driving force in protein folding.…”

Role of Ebola Virus VP30 in Transcription Reinitiation

“…This, however, only highlights the need to complement minigenome studies with the analysis of full-length genome expression systems. Recently, EBOV VP30 was shown to be an RNA-binding protein (9). Notably, VP30 binds to the sequence in the NP gene's stem-loop structure that includes the transcriptional start signal and its complementary sequence.…”

Interplay Among Constitutes of Ebola Virus: Nucleoprotein, Polymerase L, Viral Proteins

“…viral RNA suggests that the mechanism facilitating speci¯city for NP-RNA binding is related to VP30. [81][82][83] A bridge of NP-VP30-L has been suggested, which is similar to the observed NP-VP35-L bridge, indicating a complex consisting of VP30, VP35 and L that interacts with NP. 16,83 Direct interaction of VP30 with L has been shown by co-immuno°uorescence.…”

“…Direct interactions of VP30 with NP, VP35, L and RNA have all been identi¯ed, and a VP30 mediatory function of viral replication and transcription through phosphorylation shift has also been observed. 16,74,81 These¯ndings again emphasize the signi¯cance of VP30 in regulating viral replication cycle. Importantly, additional conformational changes of NP might also be induced during the process.…”

“…21,79,80 Interactions of VP30 with VP35 and RNA are related to regulation of RNA replication and transcription. 74,81 The fact that VP30 speci¯cally binds to ebola virus Initially, the N-terminal peptide of VP35 (purple ovals) binds to the NP core domain, keeping the cleft closed so that genomic ssRNA is protected from external contacts. 47 At the start of RNA synthesis, the polymerase complex comes in and interacts with VP35, and the N-peptide of VP35 becomes untapped from NP, leading to the opening of the RNA binding cleft.…”