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Mutation of K214/R260 prevents P1 nuclear accumulation. (A) COS-7 cells transfected to express the indicated proteins were treated without or with LMB (2.8 ng/ml, 3h) before imaging of living cells by CLSM and (B) determination of F n/c as described in the legend to Fig 2 (mean ± S.E.M., n ! 50 cells). Data are representative of three separate assays; statistical analysis used Student's t-test: **** p < 0.0001) doi:10.1371/journal.pone.0150477.g003
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Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isofo...
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... Similarly, nuclearlocalized influenza A virus (IAV) nucleoprotein N-terminal deletion mutants lack viral mRNA translation and exhibit defects in forming functional viral ribonucleoproteins, leading to delayed replication in IAV-infected cells [40]. In addition, recent studies have shown that nuclear import of the rabies virus P protein facilitates the inhibition of host gene transcription, regulates viral genome replication and transcription, and disrupts antiviral signaling pathways [41,42]. This result shows that NLS sequences are necessary in the virus infection process. ...
Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus’s replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4–37 of the N-terminus of the PCV4 Cap, 4RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN37. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at 4RSRYSRRRRNRRNQRR19 and 24PRASRRRYRWRRK36, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid 4RSRY7 in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs.
... The distinct localization of P3 is due to truncation of the N-NES, active nuclear import via a noncanonical N-terminal nuclear localization sequence (N-NLS, requiring sequence from residue 53, extending beyond residue 151, such that sequence encompassing residues 53 to 174 comprises activity comparable to full-length protein) (10,11), and a conformational C-terminal NLS (C-NLS, residues 211 to 214, 260). P3 also contains NES activity in the C-terminal domain (P CTD ) due to the presence of a C-NES (residues 223 to 232) (11)(12)(13), such that nucleocytoplasmic localization depends on a balance of NLS/NES activity (7). These differences in the localization of P1 and P3 appear to enable multiple mechanisms of host-cell subversion, including in immune evasion, through interactions with cellular proteins of the innate immune system (8). ...
Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.
... Residue numbering corresponds to the full-length P protein sequence. (B, C) HeLa cells were transfected to express the indicated proteins and treated with or without LMB (3 h) before imaging by live-cell CLSM at 16-20 h post-transfection (p.t), as previously.20 Yellow arrowheads indicate nucleoli. ...
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.
... Nuclear localization of P3 varies significantly between lyssaviruses. Despite the conservation of MT association between P3 of phylogroup I lyssaviruses, CLSM analysis of nuclear localization ( Fig. 3A and Fig. S4), including quantitative analysis of images to calculate the ratio of nuclear-to-cytoplasmic fluorescence (Fn/c; Fig. 3E) (44,45), indicated significant divergence between viruses. Specifically, SHBRV P3 was strongly cytoplasmic, while the P3 proteins of CVS, Ni, and EBLV2 were able to localize in the nucleus. ...
... Subcellular localization of RABV P protein has been characterized in detail, largely by using CVS P protein/isoforms, and involves multiple sequences/domains that regulate trafficking and so contribute to different STATantagonistic processes (22-25, 27, 29, 34, 44). The principal sequences regulating nuclear localization include the N-NES and N-NLS; additional regulation involves a CTD-localized NLS and NES (C-NLS and C-NES, which are present in all isoforms), while the MTAS mediates MT interaction/tethering that suppresses nuclear accumulation (Fig. 1A) (22,27,29,34,44). Furthermore, phosphorylation at S 210 is indicated to regulate nuclear localization and MT association (33,35). ...
... Image acquisition used Nikon NIS-Elements (Eclipse C1) or Leica LAS AF (SP5) software. Digitized confocal files (single slices) were analyzed using ImageJ 1.62 software (NIH) to determine the ratio of nuclear-to-cytoplasmic fluorescence (Fn/c) for each cell as previously described (44,45). The Fn/ c ratio was calculated (Fn-Fb/Fc-Fb, where F, Fb, and Fc are nuclear, background, and cytoplasmic fluorescence, respectively) to determine the relative nuclear accumulation of fluorescently labeled proteins. ...
Viral hijacking of microtubule (MT)-dependent transport is well understood, but several viruses also express discrete MT-associated proteins (vMAPs), potentially to modulate MT-dependent processes in the host cell. Specific roles for vMAP-MT interactions include subversion of antiviral responses by P3, an isoform of the P protein of rabies virus (RABV; genus Lyssavirus), which mediates MT-dependent antagonism of interferon (IFN)-dependent signal transducers and activators of transcription 1 (STAT1) signaling. P3 also undergoes nucleocytoplasmic trafficking and inhibits STAT1-DNA binding, indicative of intranuclear roles in a multipronged antagonistic strategy. MT association/STAT1 antagonist functions of P3 correlate with pathogenesis, indicating potential as therapeutic targets. However, key questions remain, including whether other P protein isoforms interact with MTs, the relationship of these interactions with pathogenesis, and the extent of conservation of P3-MT interactions between diverse pathogenic lyssaviruses. Using super-resolution microscopy, live-cell imaging, and immune signaling analyses, we find that multiple P protein isoforms associate with MTs and that association correlates with pathogenesis. Furthermore, P3 proteins from different lyssaviruses exhibit variation in intracellular localization phenotypes that are associated with STAT1 antagonist function, whereby P3-MT association is conserved among lyssaviruses of phylogroup I but not phylogroup II, while nucleocytoplasmic localization varies between P3 proteins of the same phylogroup within both phylogroup I and II. Nevertheless, the divergent P3 proteins retain significant IFN antagonist function, indicative of adaptation to favor different inhibitory mechanisms, with MT interaction important to phylogroup I viruses. IMPORTANCE Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomyelitis that is almost invariably fatal. There are no effective antivirals for symptomatic infection, and effective application of current vaccines is limited in areas of endemicity, such that rabies causes ~59,000 deaths per year. Viral subversion of host cell functions, including antiviral immunity, is critical to disease, and isoforms of the lyssavirus P protein are central to the virus-host interface underpinning immune evasion. Here, we show that specific cellular interactions of P protein isoforms involved in immune evasion vary significantly between different lyssaviruses, indicative of distinct strategies to evade immune responses. These findings highlight the diversity of the virus-host interface, an important consideration in the development of pan-lyssavirus therapeutic approaches.
... Therefore, due to the presence of regions with high intrinsic disorder content, several MoRFs, several phosphorylation sites, and the usage of alternative initiation, it is possible for the P-protein to serve many roles within the virus [10]. Furthermore, the P-protein isoforms were shown to differ in nucleocytoplasmic localization and microtubule (MT) association, mediated by several functional motifs, including the nuclear localization sequence (NLS, residues 211-214) and N-and C-terminally located nuclear export sequences (N-NES and C-NES, residues 49-58 and 223-232, respectively) [107][108][109]. For example, shorter isoforms (P3 to P5) lacking the N-terminally located NES are more nuclear and are capable of binding and bundling MTs [107]. ...
Rabies is a neurological disease that causes between 40,000 and 70,000 deaths every year. Once a rabies patient has become symptomatic, there is no effective treatment for the illness, and in unvaccinated individuals, the case-fatality rate of rabies is close to 100%. French scientists Louis Pasteur and Émile Roux developed the first vaccine for rabies in 1885. If administered before the virus reaches the brain, the modern rabies vaccine imparts long-lasting immunity to the virus and saves more than 250,000 people every year. However, the rabies virus can suppress the host’s immune response once it has entered the cells of the brain, making death likely. This study aimed to make use of disorder-based proteomics and bioinformatics to determine the potential impact that intrinsically disordered protein regions (IDPRs) in the proteome of the rabies virus might have on the infectivity and lethality of the disease. This study used the proteome of the Rabies lyssavirus (RABV) strain Pasteur Vaccins (PV), one of the best-understood strains due to its use in the first rabies vaccine, as a model. The data reported in this study are in line with the hypothesis that high levels of intrinsic disorder in the phosphoprotein (P-protein) and nucleoprotein (N-protein) allow them to participate in the creation of Negri bodies and might help this virus to suppress the antiviral immune response in the host cells. Additionally, the study suggests that there could be a link between disorder in the matrix (M) protein and the modulation of viral transcription. The disordered regions in the M-protein might have a possible role in initiating viral budding within the cell. Furthermore, we checked the prevalence of functional disorder in a set of 37 host proteins directly involved in the interaction with the RABV proteins. The hope is that these new insights will aid in the development of treatments for rabies that are effective after infection.
... Therefore, due to the presence of regions with high intrinsic disorder content, several MoRFs and several phosphorylation sites, and the usage of alternative initiation, it is possible for the P-protein to serve many roles within the virus [5]. Furthermore, the P-protein isoforms were shown to differ in nucleocytoplasmic localization and microtubule (MT) association, mediated by several functional motifs, including nuclear localization sequence (NLS, residues 211-214) and N-and C-terminally located nuclear export sequences (N-NES and C-NES, residues 49-58 and 223-232, respectively) [104][105][106]. For example, the shorter isoforms (P3 to P5) lacking the N-terminally located NES are more nuclear and are capable of binding and bundling MTs [104]. ...
Rabies is a neurological disease that causes between 40,000 and 70,000 deaths every year. Once a rabies patient has become symptomatic, there is no effective treatment for the illness, and in unvaccinated individuals, the case-fatality rate of rabies is close to 100%. French scientists Louis Pasteur and Émile Roux developed the first vaccine for rabies in 1885. If administered before the virus reaches the brain, the modern rabies vaccine imparts long-lasting immunity to the virus and saves more than 250,000 people every year. However, the rabies virus can suppress the host’s immune response once it has entered the cells of the brain, making death likely. This study aims to make use of disorder-based proteomics and bioinformatics to determine the impact that intrinsically disordered protein regions (IDPRs) in the proteome of the rabies virus have on the infectivity and lethality of the disease. This study uses the proteome of Rabies Lyssavirus (RABV) strain Pasteur Vaccins (PV), one of the best understood strains due to its use in the first rabies vaccine, as a model. The study suggests that the high levels of intrinsic disorder in the phosphoprotein (P-protein) and nucleoprotein (N-protein) allow them to participate in creation of the Negri bodies and help this virus suppress the antiviral immune response in the host cells. Additionally, the study suggests that there is a link between disorder in the matrix (M) protein and the modulation of viral transcription. The disordered regions in the M protein have a possible role in initiating viral budding within the cell. Furthermore, we checked the prevalence of functional disorder in a set of 37 host proteins directly involved in the interaction with the RABV proteins. The hope is that these new insights will aid in the development of treatments for rabies that are effective after infection.
... The P protein C-terminal domain binds directly to STAT1 but, in contrast to many other antagonists [22], does not affect STAT1 expression or tyrosine phosphorylation. Rather, P protein effects nuclear exclusion of STAT1 via a strong nuclear export sequence (N-NES) in the N-terminus of full-length P protein, and inhibits DNA interaction [8,16,17,25,30,[40][41][42][43]. Intriguingly, efficient P-protein-STAT1 interaction is dependent on IFN-treatment, such that P protein appears to antagonize pY-STAT1 selectively [16,30,32]. ...
Antagonism of the interferon (IFN)-mediated antiviral state is critical to infection by rabies virus (RABV) and other viruses, and involves interference in the IFN induction and signaling pathways in infected cells, as well as deactivation of the antiviral state in cells previously activated by IFN. The latter is required for viral spread in the host, but the precise mechanisms involved and roles in RABV pathogenesis are poorly defined. Here, we examined the capacity of attenuated and pathogenic strains of RABV that differ only in the IFN-antagonist P protein to overcome an established antiviral state. Importantly, P protein selectively targets IFN-activated phosphorylated STAT1 (pY-STAT1), providing a molecular tool to elucidate specific roles of pY-STAT1. We find that the extended antiviral state is dependent on a low level of pY-STAT1 that appears to persist at a steady state through ongoing phosphorylation/dephosphorylation cycles, following an initial IFN-induced peak. P protein of pathogenic RABV binds and progressively accumulates pY-STAT1 in inactive cytoplasmic complexes, enabling recovery of efficient viral replication over time. Thus, P protein-pY-STAT1 interaction contributes to ‘disarming’ of the antiviral state. P protein of the attenuated RABV is defective in this respect, such that replication remains suppressed over extended periods in cells pre-activated by IFN. These data provide new insights into the nature of the antiviral state, indicating key roles for residual pY-STAT1 signaling. They also elucidate mechanisms of viral deactivation of antiviral responses, including specialized functions of P protein in selective targeting and accumulation of pY-STAT1.
... Cell-based experiments showed that the P CTD contains multiple functionally important sites, including the viral N-RNA binding site (13)(14)(15) and binding sites for host factors, including nucleolin (16), microtubules (MTs) (17), promyelocytic leukemia (PML) protein (9) and signal transducers and activators of transcription (STAT) proteins (18)(19)(20). The P CTD also contains nuclear localization (C-NLS) and nuclear export (C-NES) sequences proposed to bind nuclear import (importins) and export (exportins) receptors; together with NLS/NES and other sequences in the Nterminal region, these regulate nucleocytoplasmic trafficking of P protein (21)(22)(23). The combined effect of these sequences results in a cytoplasmic distribution of P1 and P2 due to a strong N-NES that predominates over the C-NLS, with this localization presumably optimizing the cytoplasmic replication functions of P1. ...
... Further, the sequence comprising the hydrophobic residues of the C-NES that form a CRM-1 binding sequence (223 to 232, located between K214 and R260 of the C-NLS), is largely buried (8,28). Nevertheless, nucleocytoplasmic localization of P3 and the P CTD is enhanced by leptomycin B (an inhibitor of the exportin CRM1), suggesting that the C-NLS and C-NES are functional (22,23). Intriguingly, since the C-NLS is formed dependent on the P CTD fold and flanks buried residues of the C-NES, exposure of the NES to interact with CRM1 could simultaneously disable the C-NLS, efficiently switching between import and export. ...
Rabies virus P protein is a multifunctional protein with critical roles in replication and manipulation of host-cell processes, including subversion of immunity. This functional diversity involves interactions of several P protein isoforms with the cell nucleus and microtubules.
... Nuclear import of fluorescently labelled PKC-θ (DTAF-PKC-θ) was reconstituted in vitro in mechanically perforated HTC cells in the presence (+) or absence (−) of exogenous cytosol and an ATP regeneration system as previously shown [27,28]. Confocal laser scanning microscopy (CLSM) images were acquired periodically for measurement of accumulation of DTAF-PKC-θ into intact nuclei. ...
Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.
... Digitized images were processed using Fiji software (NIH). To quantify nucleocytoplasmic localization, the ratio of nuclear to cytoplasmic fluorescence, corrected for background fluorescence (Fn/c), was calculated for individual cells, before calculation of the mean Fn/c for n > 30 cells [11,[21][22][23]]. ...
... Karyopherin overexpression is a common approach to examine karyopherin-cargo interactions as it drives high levels of nuclear transport, resulting in nuclear translocation of import cargoes (e.g., [27,28]). Thus, to examine whether VP24/Kα1 complexes can accumulate within the nucleus, we analyzed COS7 cells expressing VP24, with or without co-expression of FLAG-tagged Kα1, by CLSM (Figure 1a), and calculated the nuclear to cytoplasmic fluorescence ratio (Fn/c, Figure 1b) to quantify nucleocytoplasmic localization, as previously described [11,[21][22][23].To enable analysis of VP24 localization in both fixed and living cells (below), we expressed VP24 fused to GFP (GFP-VP24), a standard approach used to characterize nuclear trafficking mechanisms of diverse proteins (e.g., [22,23,[28][29][30]). GFP fusion also increases the size of VP24 from 24 kDa (below the~40-60 kDa limit for diffusion through the NPC) to >50 kDa, and so may facilitate identification of active trafficking mechanisms. The localization of GFP-VP24 (below) was consistent with localization reported for VP24 in infected cells [6,7] and in cells expressing HA-or GFP-tagged VP24 [10,31]; GFP-VP24 was also functional for Kα1 binding and antagonism of STAT1 localization/signaling ( [11,31] and this study, below), indicating that GFP fusion is well tolerated by the protein. ...
... Karyopherin overexpression is a common approach to examine karyopherin-cargo interactions as it drives high levels of nuclear transport, resulting in nuclear translocation of import cargoes (e.g., [27,28]). Thus, to examine whether VP24/Kα1 complexes can accumulate within the nucleus, we analyzed COS7 cells expressing VP24, with or without co-expression of FLAG-tagged Kα1, by CLSM (Figure 1a), and calculated the nuclear to cytoplasmic fluorescence ratio (Fn/c, Figure 1b) to quantify nucleocytoplasmic localization, as previously described [11,[21][22][23].To enable analysis of VP24 localization in both fixed and living cells (below), we expressed VP24 fused to GFP (GFP-VP24), a standard approach used to characterize nuclear trafficking mechanisms of diverse proteins (e.g., [22,23,[28][29][30]). GFP fusion also increases the size of VP24 from 24 kDa (below the~40-60 kDa limit for diffusion through the NPC) to >50 kDa, and so may facilitate identification of active trafficking mechanisms. ...
Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.