The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.

Cytokine-induced slowing of STAT3 nuclear import; faster basal trafficking of the STAT3ß isoform.

The STAT3 signal transducer and activator of transcription is a key mediator of gene transcription in response to cytokines such as oncostatin M (OSM). We performed direct live cell imaging of GFP-tagged STAT3 proteins for the first time, showing transient relocalization of STAT3α to the nucleus following OSM exposure, in contrast to sustained nuclear relocalization of the shorter STAT3ß spliceform. To explore this further, we applied fluorescence recovery after photobleaching (FRAP) to determine the nuclear import kinetics of STAT3α and ß, as well as of a C-terminal truncation derivative STAT3ΔC comprising only the sequence shared by the spliceforms, in the absence or presence of OSM. The rates of basal nuclear import for STAT3ß and STAT3ΔC were significantly faster than those for STAT3α. Strikingly, OSM slowed the import rates of all the three STAT3 proteins, whereas the import rates of GFP alone or a classical importin-mediated cargo were unaffected, with analysis of Y705F mutant derivatives for all the three STAT3 constructs, or of a S727A mutant within the unique C-terminus of STAT3α, reinforcing the contribution of specific phosphorylation to the cytokine-stimulated changes. The results introduce a new paradigm where cytokine treatment prolongs nuclear retention simultaneous with decreasing rather than increasing the rate of nuclear import.

Oxidative stress impairs multiple regulatory events to drive persistent cytokine-stimulated STAT3 phosphorylation.

Although cytokine-driven STAT3 phosphorylation and activation are often transient, persistent activation of STAT3 is a hallmark of a range of pathologies and underpins altered transcriptional responses. As triggers in disease frequently include combined increases in inflammatory cytokine and reactive oxygen species levels, we report here how oxidative stress impacts on cytokine-driven STAT3 signal transduction events. In the model system of murine embryonic fibroblasts (MEFs), combined treatment with the interleukin-6 family cytokine Leukemia Inhibitory Factor (LIF) and hydrogen peroxide (H2O2) drove persistent STAT3 phosphorylation whereas STAT3 phosphorylation increased only transiently in response to LIF alone and was not increased by H2O2 alone. Surprisingly, increases in transcript levels of the direct STAT3 gene target SOCS3 were delayed during the combined LIF + H2O2 treatment, leading us to probe the impact of oxidative stress on STAT3 regulatory events. Indeed, LIF + H2O2 prolonged JAK activation, delayed STAT3 nuclear localisation, and caused relocalisation of nuclear STAT3 phosphatase TC-PTP (TC45) to the cytoplasm. In exploring the nuclear import/ export pathways, we observed disruption of nuclear/cytoplasmic distributions of Ran and importin-alpha3 in cells exposed to H2O2 and the resultant reduced nuclear trafficking of Classical importin-alpha/3-dependent protein cargoes. CRM1-mediated nuclear export persisted despite the oxidative stress insult, with sustained STAT3 Y705 phosphorylation enhancing STAT3 nuclear residency. Our studies thus reveal for the first time the striking impact of oxidative stress to sustain STAT3 phosphorylation and nuclear retention following disruption of multiple regulatory events, with significant implications for STAT3 function.

Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress.

The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/ß1 heterodimer and independent of importins α3, α4, ß2, ß3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress.

Tracking protein aggregation and mislocalization in cells with flow cytometry.

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival.

p32 [also known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)] has been shown previously to have both mitochondrial and non-mitochondrial localization and functions. In the present study, we show for the first time that endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions. In defining the impact of altering p32 levels in these cells, we demonstrate that the overexpression of p32 increased mitochondrial fibrils. Conversely, siRNA-mediated p32 knockdown enhanced mitochondrial fragmentation accompanied by a loss of detectable levels of the mitochondrial fusion mediator proteins Mfn (mitofusin) 1 and Mfn2. More detailed ultrastructure analysis by transmission electron microscopy revealed aberrant mitochondrial structures with less and/or fragmented cristae and reduced mitochondrial matrix density as well as more punctate ER (endoplasmic reticulum) with noticeable dissociation of their ribosomes. The analysis of mitochondrial bioenergetics showed significantly reduced capacities in basal respiration and oxidative ATP turnover following p32 depletion. Furthermore, siRNA-mediated p32 knockdown resulted in differential stress-dependent effects on cell death, with enhanced cell death observed in the presence of hyperosmotic stress or cisplatin treatment, but decreased cell death in the presence of arsenite. Taken together, our studies highlight the critical contributions of the p32 protein to the morphology of mitochondria and ER under normal cellular conditions, as well as important roles of the p32 protein in cellular metabolism and various stress responses.

Selective STAT3-α or -ß expression reveals spliceform-specific phosphorylation kinetics, nuclear retention and distinct gene expression outcomes.

Phosphorylation of STAT3 (signal transducer and activator of transcription 3) is critical for its nuclear import and transcriptional activity. Although a shorter STAT3ß spliceform was initially described as a negative regulator of STAT3α, gene knockout studies have revealed that both forms play critical roles. We have expressed STAT3α and STAT3ß at comparable levels to facilitate a direct comparison of their functional effects, and have shown their different cytokine-stimulated kinetics of phosphorylation and nuclear translocation. Notably, the sustained nuclear translocation and phosphorylation of STAT3ß following cytokine exposure contrasted with a transient nuclear translocation and phosphorylation of STAT3α. Importantly, co-expression of the spliceforms revealed that STAT3ß enhanced and prolonged the phosphorylation and nuclear retention of STAT3α, but a STAT3ß R609L mutant, with a disrupted SH2 (Src homology 2) domain, was not tyrosine phosphorylated following cytokine stimulation and could not cross-regulate STAT3α. The physiological importance of prolonged phosphorylation and nuclear retention was indicated by transcriptome profiling of STAT3(-/-) cells expressing either STAT3α or STAT3ß, revealing the complexity of genes that are up- and down-regulated by the STAT3 spliceforms, including a distinct set of STAT3ß-specific genes regulated under basal conditions and after cytokine stimulation. These results highlight STAT3ß as a significant transcriptional regulator in its own right, with additional actions to cross-regulate STAT3α phosphorylation and nuclear retention after cytokine stimulation.

Opposing actions of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) in regulating microtubule stabilization during cardiac hypertrophy.

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.

c-Jun N-terminal kinase/c-Jun inhibits fibroblast proliferation by negatively regulating the levels of stathmin/oncoprotein 18.

The JNKs (c-Jun N-terminal kinases) are stress-activated serine/threonine kinases that can regulate both cell death and cell proliferation. We have developed a cell system to control JNK re-expression at physiological levels in JNK1/2-null MEFs (murine embryonic fibroblasts). JNK re-expression restored basal and stress-activated phosphorylation of the c-Jun transcription factor and attenuated cellular proliferation with increased cells in G1/S-phase of the cell cycle. To explore JNK actions to regulate cell proliferation, we evaluated a role for the cytosolic protein, STMN (stathmin)/Op18 (oncoprotein 18). STMN, up-regulated in a range of cancer types, plays a crucial role in the control of cell division through its regulation of microtubule dynamics of the mitotic spindle. In JNK1/2-null or c-Jun-null MEFs or cells treated with c-Jun siRNA (small interfering RNA), STMN levels were significantly increased. Furthermore, a requirement for JNK/cJun signalling was demonstrated by expression of wild-type c-Jun, but not a phosphorylation-defective c-Jun mutant, being sufficient to down-regulate STMN. Critically, shRNA (small hairpin RNA)-directed STMN down-regulation in JNK1/2-null MEFs attenuated proliferation. Thus JNK/c-Jun regulation of STMN levels provides a novel pathway in regulation of cell proliferation with important implications for understanding the actions of JNK as a physiological regulator of the cell cycle and tumour suppressor protein.

The Potential Role of the ZIKV NS5 Nuclear Spherical-Shell Structures in Cell Type-Specific Host Immune Modulation during ZIKV Infection.

The Zika virus (ZIKV) non-structural protein 5 (NS5) plays multiple viral and cellular roles during infection, with its primary role in virus RNA replication taking place in the cytoplasm. However, immunofluorescence assay studies have detected the presence of ZIKV NS5 in unique spherical shell-like structures in the nuclei of infected cells, suggesting potentially important cellular roles of ZIKV NS5 in the nucleus. Hence ZIKV NS5's subcellular distribution and localization must be tightly regulated during ZIKV infection. Both ZIKV NS5 expression or ZIKV infection antagonizes type I interferon signaling, and induces a pro-inflammatory transcriptional response in a cell type-specific manner, but the mechanisms involved and the role of nuclear ZIKV NS5 in these cellular functions has not been elucidated. Intriguingly, these cells originate from the brain and placenta, which are also organs that exhibit a pro-inflammatory signature and are known sites of pathogenesis during ZIKV infection in animal models and humans. Here, we discuss the regulation of the subcellular localization of the ZIKV NS5 protein, and its putative role in the induction of an inflammatory response and the occurrence of pathology in specific organs during ZIKV infection.

Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin-α and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells.

The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barré syndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral nonstructural protein 5 (NS5), the most conserved protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies. Strikingly, ZIKV NS5 forms spherical shell-like nuclear bodies that exclude DNA. The putative monopartite NLS 390KRPR393 is necessary to direct FLAG-tagged NS5 to the nucleus as the NS5 390ARPA393 mutant protein accumulates in the cytoplasm. Furthermore, coimmunostaining experiments reveal that NS5 localizes with and sequesters importin-α, but not importin-ß, in the observed nuclear bodies during virus infection. Structural and biochemical data demonstrate binding of ZIKV NS5 with importin-α and reveal important binding determinants required for their interaction and formation of complexes that give rise to the supramolecular nuclear bodies. Significantly, we demonstrate a neuronal-specific activation of the host immune response to ZIKV infection and a possible role of ZIKV NS5's nuclear localization toward this activation. This suggests that ZIKV pathogenesis may arise from a tissue-specific host response to ZIKV infection.

Quantifying the dynamics of the oligomeric transcription factor STAT3 by pair correlation of molecular brightness.

Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. Here we present a fluorescence microscopy analysis termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture. pCOMB amplifies the signal from the brightest species present and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. We use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that on entering the nucleus STAT3 dimers must first bind DNA to form STAT3 tetramers, which are also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross-pair correlation analysis (cpCOMB) reveals that chromatin accessibility modulates STAT3 tetramer formation. Thus, the pCOMB approach is suitable for mapping the impact oligomerization on transcription factor dynamics.

Dual role of Src kinase in governing neuronal survival.

BACKGROUND: Src-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear. OBJECTIVE: To define the effect of inhibition of Src activity and expression on neuronal survival. RESULTS: In agreement with our previous findings, we demonstrated that Src was cleaved by calpain to form a 52-kDa truncated fragment in neurons undergoing excitotoxic cell death, and expression of the recombinant truncated Src fragment induced neuronal death. The data confirm that the neurotoxic signaling pathways are intact in the neurons we used for our study. To define the functional role of neuronal SFKs, we treated these neurons with SFK inhibitors and discovered that the treatment induced cell death, suggesting that the catalytic activity of one or more of the neuronal SFKs is critical to neuronal survival. Using small hairpin RNAs that suppress Src expression, we demonstrated that Src is indispensable to neuronal survival. Additionally, we found that neuronal death induced by expression of the neurotoxic truncated Src mutant, treatment of SFK inhibitors or knock-down of Src expression caused inhibition of the neuroprotective protein kinases Erk1/2, or Akt. CONCLUSIONS: Src is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases.

Hyperosmotic stress sustains cytokine-stimulated phosphorylation of STAT3, but slows its nuclear trafficking and impairs STAT3-dependent transcription.

Persistent STAT3 phosphorylation and nuclear retention are hallmarks of a range of pathologies suggesting the importance of STAT3 transcriptional responses in disease progression. Since hyperosmotic stress (HOS) is a hallmark of diseases such as diabetes and asthma, we analysed the impact of HOS on cytokine-stimulated STAT3 signalling. In contrast to transient STAT3 Y705 and S727 phosphorylation in murine embryonic fibroblasts (MEFs) stimulated by the interleukin-6 family cytokine, leukemia inhibitory factor (LIF), under non-stress conditions, HOS induced by sorbitol treatment increased STAT3 S727 but not Y705 phosphorylation. Strikingly, combined LIF+HOS treatment stimulated persistent STAT3 Y705 and S727 phosphorylation and nuclear localisation, but STAT3 nuclear accumulation was slowed during HOS, likely reflecting the mislocalisation of Ran and importin-α3 during HOS that also reduced the nuclear localisation of classical importin-α/ß-recognised nuclear import cargoes. Strikingly, combined LIF+HOS exposure, even though stimulating STAT3 phosphorylation and nuclear accumulation did not elicit a transcriptional output, as demonstrated by qPCR analyses of its target genes SOCS3 and c-Fos. Our analysis thus shows for the first time that HOS can disconnect nuclear, phosphorylated STAT3 from transcriptional outcomes, and emphasizes the importance of assessing STAT3 target gene changes in addition to STAT3 phosphorylation status and localisation.