Basic Residues within the Ebolavirus VP35 Protein Are Required for Its Viral Polymerase Cofactor Function

Abstract

The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-␣/␤) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

Predictive and comparative analysis of Ebolavirus proteins

“…17,18 VP30 is a transcription anti-terminator 19,20 and regulates the switch between transcription and replication. 21,22 VP35 acts as a cofactor of the polymerase, 23,24 and VP40 may also play a role in genome replication and transcription. 25 VP24 and VP35 participate in viral nucleocapsid assembly, 18 and VP40 is essential for virus budding and assembly.…”

Structural basis for Marburg virus VP35–mediated immune evasion mechanisms

“…Despite overall similarities in genome size and organization, virion structure, and disease characteristics (3), EBOV and MARV exhibit important differences, including their strategies for immune evasion (4). For example, although EBOV viral protein (VP) 24 and MARV VP40 counter IFN signaling, neither MARV VP24 nor EBOV VP40 appears to function similarly to its corresponding counterparts with regard to immune evasion (5)(6)(7)(8)(9)(10).…”

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

“…28,75 The¯rst basic patch (VP35 residues K222, R225, K248 and K251) in the VP35 IID is essential for NP binding by coimmunoprecipitation experiment. 64 NP and VP35 as components are important for NC morphology, and the NP: VP35 ratio is shown to modulate NC structures in vitro À À À too much VP35 (at a VP35 to NP ratio of 2.5:1) distorts NC helices, suggested by immuno°uorescence analysis. 76 The distortion induced by excessive VP35 proteins to the NC structure indicates that NC assembly is modi¯ed by the ratio of VP35 to NP, indicating that VP35 likely binds to NP in a complete NC structure and is able to modify the NC conformation by altering the binding ratio of VP35 to NP (Fig.…”

“…The domains for VP35 binding to NP and L, respectively, have been identi¯ed by Western blotting of anti-HA pull downs for VP35 and NP: the N-terminal domain of VP35 (residues 1-80) speci¯cally binds with NP, whereas the C-terminal domain of VP35 (residues 221-340) interacts with both NP and L. 15,64 Therefore, VP35 possibly serves similar roles to its counterpart P protein in vesicular stomatitis virus (VSV). In VSV, the dimer of the P protein recruits VSV L to the NC and releases partially the genomic RNA permitting subsequent RNA synthesis.…”

“…The VP35 dimer facilitates the partial release of viral ssRNA from the NP-RNA complex to the polymerase and stabilizes the intermediate dsRNA structure during RNA synthesis so that the dsRNA is protected from host immune responses. 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.…”