Phosphorylation of Ebola Virus VP30 Influences the Composition of the Viral Nucleocapsid Complex

Biedenkopf, Nadine; Hartlieb, Bettina; Hoenen, Thomas; Becker, Stephan

doi:10.1074/jbc.m113.461285

Cited by 99 publications

(143 citation statements)

References 33 publications

(53 reference statements)

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“…The only other mononegavirales protein with known similar function to M2-1 is EBOV VP30. Both M2-1 and VP30 influence transcription, interact with the viral RNA synthesis machinery, possess ZBDs, and are dynamically phosphorylated (15,16,20). VP30 displays functional similarities with M2-1, and previous structural alignment of the VP30 C-terminal domain (21) with the truncated M2-1 NMR structure revealed significant structural similarities (12).…”

Section: Discussionmentioning

confidence: 99%

“…The structural similarity between these proteins implies that they may act via common mechanisms. Recently, the role of phosphorylation in VP30 activity was probed using phosphomimetic (DD) and phosphoablatant (AA) mutants using a minigenome (16). VP30-DD stimulated replication by almost twofold and similarly diminished transcription.…”

Section: Discussionmentioning

confidence: 99%

“…Based on mutational analysis and a partial M2-1 structure determined using NMR (12,13), M2-1 is predicted to comprise four functionally significant regions: an N-terminal Cys 3 -His 1 zinc-binding domain (ZBD) (14); an alpha-helical region proposed to mediate oligomerization (11); the "core" domain (residues ∼58-177) assigned to RNA-and P-binding; and an unstructured C terminus. The core exhibits significant structural homology with the Ebola virus (EBOV) VP30 transcription factor (12), which interestingly also possesses an N-terminal zinc-binding domain (15) and phosphorylation sites that regulate VP30 function (16).…”

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confidence: 99%

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Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

Tanner

Ariza

Richard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

131

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The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.H uman respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children and the immunocompromised. HRSV is a pneumovirus of the Paramyxoviridae family of the order Mononegavirales-the nonsegmented negative-strand RNA viruses. Its genome encodes 10 genes that are each transcribed by an RNA-dependant RNA polymerase (RdRp) into single mRNAs. During transcription, the RdRp uses a single promoter in the 3′ leader region (Le) of the genome (1) and responds to gene start and gene end sequences flanking each gene, directing initiation and termination of mRNA transcription, respectively (2). During genome replication, the RdRp bypasses these signals to synthesize a full-length antigenome. The virus-encoded components needed for RNA replication are the large protein (L), the nucleocapsid protein (N), and the phosphoprotein (P). However, complete transcription of mRNAs also requires the M2-1 transcription antiterminator protein (3, 4).M2-1 prevents premature transcription termination both intra-and intergenically (5, 6). M2-1 is essential for HRSV multiplication although it is not currently known how M2-1 effects its role, and deciphering this role is complicated by its multiple interactions with other viral components, namely P (7, 8), RNA (9), and the matrix protein (M) (10). M2-1 is a 194 amino acid, basic protein that forms a stable tetramer in solution (11). Based on mutational analysis and a partial M2-1 structure determined using NMR (12, 13), M2-1 is predicted to comprise four functionally significant regions: an N-terminal Cys 3 -His 1 zinc-binding domain (ZBD) (14); an alpha-helical region proposed to mediate oligomerization (11); the "core" domain (residues ∼58-177) assigned to RNA-and P-binding; and an unstructured C terminus. The core exh...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

Tanner

Ariza

Richard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

131

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Predictive and comparative analysis of Ebolavirus proteins

2015

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Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus.

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“…VP30 allows the polymerase to read beyond a cis-RNA element in the NP mRNA 5 0 untranslated region (Weik et al, 2002) and to re-initiate transcription at gene junctions (Martínez et al, 2008). Furthermore, VP30 phosphorylation modulates the composition and function of the RNA synthesis machinery (Biedenkopf et al, 2013;Martinez et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Structure of theReston ebolavirusVP30 C-terminal domain

Clifton¹,

Kirchdoerfer

Atkins³

et al. 2014

Acta Cryst Sect F

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PDB reference: Reston ebolavirus VP30 C-terminal domain, 3v7oThe ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.

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Phosphorylation of Ebola Virus VP30 Influences the Composition of the Viral Nucleocapsid Complex

Cited by 99 publications

References 33 publications

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

Predictive and comparative analysis of Ebolavirus proteins

Structure of theReston ebolavirusVP30 C-terminal domain

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