Structural comparison of the C-terminal domain of functionally divergent lyssavirus P proteins

Sugiyama, Aoi; Tomo, Nomai; Jiang, Xue; Minami, Miku; Yao, Min; Maenaka, Katsumi; Ito, Naoto; Gooley, Paul R.; Moseley, Gregory W.; Ose, Toyoyuki

doi:10.1016/j.bbrc.2020.05.195

Cited by 5 publications

(6 citation statements)

References 40 publications

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“…Nucleolar localization signals (NoLSs) are highly variable and typically poorly defined, but they often overlap with NLSs, 38 so we initially examined whether residues K214/R260, which form part of a previously defined NLS in the CTD (C-NLS), affect nucleolar targeting. [39][40][41][42] Analysis of GFP-fused P3 in HeLa cells by live-cell CLSM confirmed previous observations 20 that wild-type (wt) P3 localises in the nucleus and cytoplasm, and can accumulate in nucleoli (Figure 4B). In contrast to henipavirus M proteins, no strong accumulation into FC-DFC was observed, with P3 having a more diffuse appearance throughout the nucleolus 11 (Figure 4D).…”

Section: Mutation Of Residues K214-a and R260-a (Krm) In Rabv P3 Abol...supporting

confidence: 88%

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

Rawlinson

Zhao

Ardipradja

et al. 2022

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The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically‐replicating negative‐sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA‐damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub‐nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.

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Section: Mutation Of Residues K214-a and R260-a (Krm) In Rabv P3 Abol...supporting

confidence: 88%

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

Rawlinson

Zhao

Ardipradja

et al. 2022

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“…Analysis of the chemical shift perturbations for W265 and C271 of this W-hole is consistent with the apparent K D calculated for a single-site interaction and are likely to reflect the interaction with the positive patch. These data are in agreement with a lack of support for roles of the W-hole residues in mutagenic studies of N binding [26,32]; notably the W-hole residues, especially W265, are poorly conserved among lyssaviruses [27] (S6B Fig) . The original proposal that the W-hole might be involved in P CTD binding [26] was based on the presence of mutations in the region corresponding to the W-hole in MOKV P CTD constructs (in MOKV the W is substituted for F) that were defective for N binding in a yeast two-hybrid random mutagenesis screen [32]. As all these constructs also contained mutations to the conserved positive patch, it seems likely that the lack of N binding was independent of mutations in the W-hole.…”

Section: Discussionsupporting

confidence: 75%

“…Cellular interactors include critical components of interferon (IFN) signalling pathways, and several elements of the host cellular trafficking machinery [22,23], which enable P protein to travel between the host cell cytoplasm and nucleus, a process implicated in immune evasion [18,24,25]. The well-structured C-terminal globular domain (P CTD ), spanning residues 186-297 of P protein [26,27], represents a critical interface for viral replication as the site of interaction with N-RNA [14,28], as well as for interactions with host-cell proteins, including several signal transducers and activators of transcription (STAT) proteins, important to immune evasion [16][17][18]20,21,24,29].…”

Section: Introductionmentioning

confidence: 99%

“…However, these predictions are controversial as there are no direct data supporting a role for the W-hole in N binding [31]. Importantly, as the residues of the W-hole are not conserved, the potential interaction surface would differ for P proteins of different strains, casting doubt that the Whole forms a common interaction site [27]. Furthermore, while yeast two-hybrid mutagenic analysis performed with MOKV proteins [31] and mammalian cell-based assays performed with RABV proteins [29] were consistent with a role for the positive patch, the latter study suggested that mutations of the W-hole residues did not impair N-binding or viral replication.…”

Section: Introductionmentioning

confidence: 99%

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Definition of the immune evasion-replication interface of rabies virus P protein

et al. 2021

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Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a ‘positive patch’ of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.

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“…The P protein is a multifunctional protein, consisting of a long-disordered N-terminal domain (amino acid 1-90), a central dimerization domain (DD, amino acid 91-133), a disordered linker (amino acid 134-185) and a C-terminal domain (CTD, amino acid 185-297), containing a large number of host as well as viral protein binding sites [23]. Amino acids at positions 1-60 of the P protein bind to the N protein, which prevents the N protein from binding to host nonspecific RNA [24], and amino acids at positions 11-50 of the P protein activate the L protein [25,26].…”

Section: The Pathogenic Mechanisms Of Rabv P Proteinmentioning

confidence: 99%

Research Advances on the Interactions between Rabies Virus Structural Proteins and Host Target Cells: Accrued Knowledge from the Application of Reverse Genetics Systems

Yin

Wang

Mao

et al. 2021

Viruses

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Rabies is a lethal zoonotic disease caused by lyssaviruses, such as rabies virus (RABV), that results in nearly 100% mortality once clinical symptoms appear. There are no curable drugs available yet. RABV contains five structural proteins that play an important role in viral replication, transcription, infection, and immune escape mechanisms. In the past decade, progress has been made in research on the pathogenicity of RABV, which plays an important role in the creation of new recombinant RABV vaccines by reverse genetic manipulation. Here, we review the latest advances on the interaction between RABV proteins in the infected host and the applied development of rabies vaccines by using a fully operational RABV reverse genetics system. This article provides a background for more in-depth research on the pathogenic mechanism of RABV and the development of therapeutic drugs and new biologics.

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Structural comparison of the C-terminal domain of functionally divergent lyssavirus P proteins

Cited by 5 publications

References 40 publications

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

Definition of the immune evasion-replication interface of rabies virus P protein

Research Advances on the Interactions between Rabies Virus Structural Proteins and Host Target Cells: Accrued Knowledge from the Application of Reverse Genetics Systems

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