Phosphorylation Of Nucleoprotein Research Articles

PLK3 facilitates replication of swine influenza virus by phosphorylating viral NP protein

ABSTRACT Swine H1N1/2009 influenza is a highly infectious respiratory disease in pigs, which poses a great threat to pig production and human health. In this study, we investigated the global expression profiling of swine-encoded genes in response to swine H1N1/2009 influenza A virus (SIV-H1N1/2009) in newborn pig trachea (NPTr) cells. In total, 166 genes were found to be differentially expressed (DE) according to the gene microarray. After analyzing the DE genes which might affect the SIV-H1N1/2009 replication, we focused on polo-like kinase 3 (PLK3). PLK3 is a member of the PLK family, which is a highly conserved serine/threonine kinase in eukaryotes and well known for its role in the regulation of cell cycle and cell division. We validated that the expression of PLK3 was upregulated after SIV-H1N1/2009 infection. Additionally, PLK3 was found to interact with viral nucleoprotein (NP), significantly increased NP phosphorylation and oligomerization, and promoted viral ribonucleoprotein assembly and replication. Furthermore, we identified serine 482 (S482) as the phosphorylated residue on NP by PLK3. The phosphorylation of S482 regulated NP oligomerization, viral polymerase activity and growth. Our findings provide further insights for understanding the replication of influenza A virus.

Nucleoprotein phosphorylation site (Y385) mutation confers temperature sensitivity to influenza A virus due to impaired nucleoprotein oligomerization at a lower temperature.

Mutations in viral proteins can lead to the cold adaption of influenza A virus and the cold-adapted virus is an important vaccination instrument. Here, we identify a novel strain of influenza A virus with cold sensitivity conferred by a mutation at a phosphorylation site within the nucleoprotein (NP). The highly conserved tyrosine 385 residue (Y385) of NP was identified as a phosphorylation site by mass spectrometry. The constructive NP phosphorylation mimicked by Y385E mutation was fatal for virus replication, while the continuous Y385 dephosphorylation mimicked by Y385F mutation had little impact on virus replication in vitro. Notably, the Y385F virus showed much lower replicative capacity in turbinates of mice compared with the wild type virus. Moreover, the replication of Y385F virus was significantly reduced in both A549 and MDCK cells grown at 33°C, when compared to that at 37°C. These results indicated that the Y385F mutation led to cold sensitivity of virus. We further found that the cold sensitivity of Y385F virus could be attributed to diminished NP oligomerization rather than any changes in intracellular localization. Taken together, these findings suggest that the phosphorylation of NP may be a critical factor that regulates the temperature sensitivity of influenza A virus.

Phosphorylation Status of Tyrosine 78 Residue Regulates the Nuclear Export and Ubiquitination of Influenza A Virus Nucleoprotein

Phosphorylation and dephosphorylation of nucleoprotein (NP) play significant roles in the life cycle of influenza A virus (IAV), and the biological functions of each phosphorylation site on NP are not exactly the same in controlling viral replication. Here, we identified tyrosine 78 residue (Y78) of NP as a novel phosphorylation site by mass spectrometry. Y78 is highly conserved, and the constant NP phosphorylation mimicked by Y78E delayed NP nuclear export through reducing the binding of NP to the cellular export receptor CRM1, and impaired virus growth. Furthermore, the tyrosine kinase inhibitors Dasatinib and AG490 reduced Y78 phosphorylation and accelerated NP nuclear export, suggesting that the Janus and Src kinases-catalyzed Y78 phosphorylation regulated NP nuclear export during viral replication. More importantly, we found that the NP phosphorylation could suppress NP ubiquitination via weakening the interaction between NP and E3 ubiquitin ligase TRIM22, which demonstrated a cross-talk between the phosphorylation and ubiquitination of NP. This study suggests that the phosphorylation status of Y78 regulates IAV replication by inhibiting the nuclear export and ubiquitination of NP. Overall, these findings shed new light on the biological roles of NP phosphorylation, especially its negative role in NP ubiquitination.

CDC25B promotes influenza A virus replication by regulating the phosphorylation of nucleoprotein

Cell division cycle 25 B (CDC25B) is a member of the CDC25 phosphatase family. It can dephosphorylate cyclin-dependent kinases and regulate the cell division cycle. Moreover, siRNA knockdown of CDC25B impairs influenza A virus (IAV) replication. Here, to further understand the regulatory mechanism of CDC25B for IAV replication, a CDC25B-knockout (KO) 293T cell line was constructed using CRISPR/Cas9. The present data indicated that the replication of IAV was decreased in CDC25B-KO cells. Additionally, CDC25B deficiency damaged viral polymerase activity, nucleoprotein (NP) self-oligomerization, and NP nuclear export. Most importantly, we found that the NP phosphorylation levels were significantly increased in CDC25B-KO cells. These findings indicate that CDC25B facilitates the dephosphorylation of NP, which is vital for regulating NP functions and the life cycle of IAV.

Phosphorylation and dephosphorylation of threonine 188 in nucleoprotein is crucial for the replication of influenza A virus

Nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex that is responsible for viral replication, transcription and packaging of influenza A virus. Phosphorylation of NP plays an important role during viral infection. In the present study, we identified threonine 188 (T188) as a novel phosphorylated residue in the NP of influenza A virus by using mass spectrometry. T188 is located within nuclear export signal 2 (NES2) which is chromosome region maintenance 1 (CRM1)-independent. We observed that the phosphorylation and dephosphorylation of residue T188 regulated viral replication by controlling NES2-dependent NP nuclear export and the polymerase activity of the vRNP complex. Our findings provide further insights for understanding the replication of influenza A virus.

Demonstration of the Receptor Site for Thyroid Hormone on Integrin αvβ3.

Integrin αvβ3 is one of the 24 heterodimeric structural proteins of the plasma membrane of animal cells. The extracellular domain of αvβ3 comprises about 80% of its mass and is devoted largely to interactions with extracellular matrix (ECM) proteins, such as vitronectin and fibronectin, and cell-cell interaction. The binding of specific ECM proteins is conveyed to the cell interior by signal transduction. In the past decade, integrin αvβ3 has been shown to bind small molecules, such as nonprotein hormones-thyroid hormone analogues and dihydrotestosterone-and the stilbene, resveratrol. The αvβ3-thyroid hormone interaction signal generated at the integrin is transduced into intracellular protein trafficking, nucleoprotein phosphorylation, and expression of specific genes. The hormone receptor on the integrin bears no structural homologies with nuclear thyroid hormone receptors (TRs). Because integrin αvβ3 is generously expressed on the surface of cancer cells and rapidly dividing endothelial cells, thyroid hormone actions initiated at the receptor on integrin not unexpectedly relate to cell proliferation, cancer cell survival pathways and to angiogenesis. In this chapter, we present methods for the definition of the receptor, for monitoring certain of its functions and for the downregulation of the gene for the integrin.

Phosphorylation at the homotypic interface regulates nucleoprotein oligomerization and assembly of the influenza virus replication machinery.

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle.

Phosphorylation controls the nuclear-cytoplasmic shuttling of influenza A virus nucleoprotein.

The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-α. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication. It is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.

Cancer Cell Gene Expression Modulated from Plasma Membrane Integrin αvβ3 by Thyroid Hormone and Nanoparticulate Tetrac.

Integrin αvβ3 is generously expressed by cancer cells and rapidly dividing endothelial cells. The principal ligands of the integrin are extracellular matrix proteins, but we have described a cell surface small molecule receptor on αvβ3 that specifically binds thyroid hormone and thyroid hormone analogs. From this receptor, thyroid hormone (l-thyroxine, T4; 3,5,3′-triiodo-l-thyronine, T3) and tetraiodothyroacetic acid (tetrac) regulate expression of specific genes by a mechanism that is initiated non-genomically. At the integrin, T4 and T3 at physiological concentrations are pro-angiogenic by multiple mechanisms that include gene expression, and T4 supports tumor cell proliferation. Tetrac blocks the transcriptional activities directed by T4 and T3 at αvβ3, but, independently of T4 and T3, tetrac modulates transcription of cancer cell genes that are important to cell survival pathways, control of the cell cycle, angiogenesis, apoptosis, cell export of chemotherapeutic agents, and repair of double-strand DNA breaks. We have covalently bound tetrac to a 200 nm biodegradable nanoparticle that prohibits cell entry of tetrac and limits its action to the hormone receptor on the extracellular domain of plasma membrane αvβ3. This reformulation has greater potency than unmodified tetrac at the integrin and affects a broader range of cancer-relevant genes. In addition to these actions on intra-cellular kinase-mediated regulation of gene expression, hormone analogs at αvβ3 have additional effects on intra-cellular protein-trafficking (cytosol compartment to nucleus), nucleoprotein phosphorylation, and generation of nuclear coactivator complexes that are relevant to traditional genomic actions of T3. Thus, previously unrecognized cell surface-initiated actions of thyroid hormone and tetrac formulations at αvβ3 offer opportunities to regulate angiogenesis and multiple aspects of cancer cell behavior.

Phosphorylation of Measles Virus Nucleoprotein Affects Viral Growth by Changing Gene Expression and Genomic RNA Stability

The measles virus (MV) nucleoprotein associates with the viral RNA genome to form the N-RNA complex, providing a template for viral RNA synthesis. In our previous study, major phosphorylation sites of the nucleoprotein were identified as S479 and S510. However, the functions of these phosphorylation sites have not been clarified. In this study, we rescued recombinant MVs (rMVs) whose phosphorylation sites in the nucleoprotein were substituted (rMV-S479A, rMV-S510A, and rMV-S479A/S510A) by reverse genetics and used them in subsequent analyses. In a one-step growth experiment, rMVs showed rapid growth kinetics compared with wild-type MV, although the peak titer of the wild-type MV was the same as or slightly higher than those of the rMVs. Time course analysis of nucleoprotein accumulation also revealed that viral gene expression of rMV was enhanced during the early phase of infection. These findings suggest that nucleoprotein phosphorylation has an important role in controlling viral growth rate through the regulation of viral gene expression. Conversely, multistep growth curves revealed that nucleoprotein-phosphorylation intensity inversely correlated with viral titer at the plateau phase. Additionally, the phosphorylation intensity of the wild-type nucleoprotein in infected cells was significantly reduced through nucleoprotein-phosphoprotein binding. Excessive nucleoprotein-phosphorylation resulted in lower stability against RNase and faster turnover of viral genomic RNA. These results suggest that nucleoprotein-phosphorylation is also involved in viral genomic RNA stability.

Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75.

The influenza A virus nucleoprotein (NP) is a phosphoprotein that encapsidates the viral genomic RNA. To map the in vivo phosphorylation site(s) of this protein, 32P-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chromatography. The purified protein was then subjected to chemical digestion with formic acid, which cleaves proteins at Asp-Pro bonds, and the resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of the phosphorylated products obtained were identified as fragments corresponding to the N-terminal 88 amino acids and to the C-terminal 196 residues of the NP. To identify the phosphate acceptor site(s) at the N-terminal phosphorylated region of NP, each of the seven serines within this region was individually changed to alanine by site-directed mutagenesis. The mutant proteins were then transiently expressed in mammalian cells and analyzed for their phosphorylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of 32P label incorporated into NP, whereas the other substitutions did not have a discernible effect on the phosphorylation level of the protein. In addition, all serine-altered proteins were tested for their functionality in an artificial system in which expression of a synthetic chloramphenicol acetyl-transferase RNA molecule is driven by influenza virus proteins synthesized from cloned genes. The results obtained demonstrate that all mutant proteins were competent to cooperate with the subunits of the viral polymerase for expression of the synthetic virus-like chloramphenicol acetyltransferase RNA in vivo. These data are discussed regarding the possible roles of NP phosphorylation for the viral replicative cycle.

Phosphorylation of the Nucleoprotein of an Avian Influenza Virus

High resolution polyacrylamide gel electrophoresis (PAGE) of chick embryo fibroblast cells infected with the avian influenza virus FPV-Rostock revealed two distinct polypeptides migrating in the region of the nucleoprotein (NP). One-dimensional fingerprinting of these polypeptides showed that they were both nucleoprotein, and [32P]orthophosphate labelling revealed that they differed with respect to their state of phosphorylation. Pulse-chase studies using [35S]methionine indicated that phosphorylation of a certain proportion of NP occurs rapidly after synthesis and is associated with transport to the nucleus. Nucleoprotein which remained in the cytoplasm was predominantly non-phosphorylated. Both the phosphorylated and the non-phosphorylated types of NP were found in ribonucleoprotein complexes (RNPs) of different densities isolated on renografin gradients, but RNPs isolated from the nucleus contained much more phosphorylated NP than those from the cytoplasm. The kinase responsible for nucleoprotein phosphorylation appears to be influenced by temperature of incubation of the infected cells.

The structure and synthesis of influenza virus phosphoproteins.

The synthesis and phosphorylation of influenza virus nucleoprotein and nonstructural protein were analyzed. The nucleoprotein (NP) was found to be phosphorylated in both infected cells and in isolated virions. The phosphate is in a monoester linkage to a serine residue. Two-dimensional tryptic peptide maps of the 32P-labeled protein, as well as measurements of specific activity, suggests that NP is phosphorylated at one site per molecule. The viral nonstructural (NS 1) protein is also phosphorylated, but on threonine residues. Up to a maximum of two sites per NS 1 molecule could be so modified in infected cells, as demonstrated by two different methods of tryptic peptide analysis and by measurements of the ratio of 32P to 3H-amino-acids incorporated into NS 1 protein species. The NS 1 protein is resolved into four major species of differing isoelectric point in a two-dimensional electrophoretogram. The most acidic species was found to have two phosphorylated sites per molecule, and the next most acidic species contained on the average one phosphate per molecule. Treatment of the phosphorylated species with bacterial alkaline phosphatase demonstrated that the level of phosphorylation is the only identifiable difference between the phosphorylated and unphosphorylated NS 1 species. The distinction between the two unphosphorylated species could not be determined. The distribution of the un-, mono-, and diphosphorylated NS 1 species was characterized at different times after synthesis. These modifications were found to occur very rapidly after translation (30 to 60 s), after transport of the unmodified species from cytoplasm to nucleus of the infected cell. The phosphorylation of NP also takes place rapidly after its synthesis; the site within the cell of the NP phosphorylation has not been unambiguously determined.

Phosphorylation of myocardial nuclei enhances their susceptibility to deoxyribonuclease I

Phosphorylation of myocardial nuclei by endogenous protein kinases enhances both in vitro RNA synthesis and susceptibility to DNase I digestion. In addition, the relative proportion of a transcriptionally active chromatin fraction is higher in phsphorylated nuclei. Nucleoprotein phosphorylation may, thus, affect transcription through changes in chromatin organization.

Hormone-Induced Alterations in Nonhistone Protein: Methylation and Phosphorylation in Sciara coprophila

Methylation and phosphorylation of chromosomal nonhistone protein (NHP) has been demonstrated in the salivary gland cells of diptera [5, 7] and implicated in the control of gene expression [35, 36]. Furthermore, hormones can stimulate methyl and phosphoryl side chain metabolism and thus enhance template activity. Salivary glands from late fourth instar female larvae of Sciara coprophila (cortisone-supplemented and normal diet) were incubated in 3H-uridine (10 muCi/ml), 3H-thymidine (10 muCi/ml), 3H-methyl-methionine (20 muCi/ml), 35S-methionine (10 muCi/ml) and 32P-orthophosphate (1 mc/ml), for varying time periods, to measure RNA synthesis, DNA synthesis, methylation, protein synthesis and phosphorylation, respectively. Following selective extraction of lipid, histone and nucleic acids, glands were prepared for light microscope autoradiography. A more specific labelling pattern, as well as increased grain number on particular bands, interbands and bulbs, was noted on chromosomes from cortisone-fed larvae incubated in 3H-methyl-methionine for 1 min when compared with larvae on the standard diet. Cortisone also increased RNA synthesis and nucleoprotein phosphorylation, but not DNA or protein synthesis. In summary, cortisone enhances the specificity and degree of NHP methylation and phosphorylation at discrete chromosomal loci, i.e. alterations in side chain metabolism which may be responsible for increased RNA synthesis.

Protein phosphokinases of chick muscle: Changes during embryonic development

Protein phosphorylation is a widespread phenomenom [l] involving, among others, such distinct cell functions as enzyme conversions [2] , changes ’ in membrane permeability [3], and regulation of RNA transcription [4]. Protein phosphokinases with different substrate specificity have been found [5,6] and histone phosphorylation has been particularly implicated in gene activation through increased template activity [7]. In fact, the increased rate of nucleoprotein phosphorylation is concomitant with an active cell division like that occurring e.g. in regenerating liver [ 81 , in unilaterally nephrectomized kidney and fast growing tumoral ascites cells [9] , and the developing mammary gland [lo]. Protein kinase from rabbit muscle phosphorylates several proteins, including glycogen synthetase [ 5, 1 l] . Since the pattern during embryonic development of chick muscle glycogen synthetase kinase has been described [ 121, it was interesting to know the patterns with the other substrates. In fact, eventual differences would indicate that protein kinases with changing functions might be present during muscle differentiation. It is the purpose of this communication to report