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...
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