34 research outputs found
Comparative analysis of seven viral nuclear export signals (NESs) reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP) in influenza A virus replication.
The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication
Leukemia inhibitory factor-induced phosphorylation of STAP-2 on tyrosine-250 is involved in its STAT3-enhancing activity.
Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates their signaling pathways. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by v-src and Jak2, using a phospho-specific antibody against STAP-2 phosphorylated at Tyr250. Mutational analyses revealed that Tyr250 was involved in the STAT3-enhancing activity of STAP-2. We further found that leukemia inhibitory factor (LIF) stimulated STAP-2 Tyr250 phosphorylation in 293T and Hep3B cells. Moreover, endogenous STAP-2 was phosphorylated at Tyr250 following LIF stimulation of murine M1 cell line. Taken together, our findings demonstrate that endogenous STAP-2 is phosphorylated at Tyr250 and that this phosphorylation is involved in its function
Correction: Importin α3/Qip1 Is Involved in Multiplication of Mutant Influenza Virus with Alanine Mutation at Amino Acid 9 Independently of Nuclear Transport Function.
STAP-2 regulates c-Fms/M-CSF receptor signaling in murine macrophage Raw 264.7 cells.
Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. Our previous studies also revealed that STAP-2 binds to MyD88 and IKK-α/β, and modulates NF-κB signaling in macrophages. In the present study, we examined physiological roles of the interaction between STAP-2 and c-Fms in Raw 264.7 macrophage cells. Our immunoprecipitation has revealed that c-Fms directly interacts with the PH domain of STAP-2 independently on M-CSF-stimulation. Ectopic expression of STAP-2 markedly suppressed M-CSF-induced tyrosine phosphorylation of c-Fms as well as activation of Akt and extracellular signal regulated kinase. In addition, Raw 264.7 cells over-expressing STAP-2 showed impaired migration in response to M-CSF and wound-healing process. Taken together, our findings demonstrate that STAP-2 directly binds to c-Fms and interferes with the PI3K signaling, which leads to macrophage motility, in Raw 264.7 cells
Correction: Importin α3/Qip1 Is Involved in Multiplication of Mutant Influenza Virus with Alanine Mutation at Amino Acid 9 Independently of Nuclear Transport Function
Importin α3/Qip1 is involved in multiplication of mutant influenza virus with alanine mutation at amino acid 9 independently of nuclear transport function.
The nucleoprotein (NP) of influenza A virus is transported into the nucleus via the classical importin α/β pathway, and proceeds via nuclear localization signals (NLSs) recognized by importin α molecules. Although NP binds to importin α isoforms Rch1, Qip1 and NPI-1, the role of each individual isoform during the nuclear transport of NP and replication of the influenza virus remains unknown. In this study, we examined the contribution of importin α isoforms for nuclear localization of NP and viral growth using a panel of NP mutants containing serial alanine replacements within an unconventional NLS of NP. Alanine mutation at amino acid 8 (R8A) caused a significant reduction in the nuclear localization and binding to the three importin isoforms. The R8A NP mutant virus did not generate by reverse-genetics approach. This indicates that position 8 is the main site that mediates nuclear localization via interactions with Rch1, Qip1 and NPI-1, and subsequent viral production. This was confirmed by the finding that the conservation of amino acid 8 in human- and avian-origin influenza virus NP was necessary for virus propagation. By contrast, another mutant, S9A NP, which localized in the nucleus, caused a reduction in viral growth and vRNA transcription, suggesting that the unconventional NLS within NP may be associated with nuclear transport, vRNA transcription and viral replication through independent pathways. Interestingly, the N-terminal 110-amino acid region, which contained the unconventional NLS with S9A mutation, mainly bound to Qip1. Furthermore, activities of vRNA transcription and replication of S9A NP mutants were decreased by silencing Qip1 in without changing nuclear localization, indicating that Qip1 involves in multiplication of S9A mutant virus independently of nuclear transport function. Collectively, our results demonstrate the unconventional NLS within NP might have the additional ability to regulate the viral replication that is independent of nuclear localization activity via interactions with Qip1
Summary of viral replication kinetics, nuclear localization, nuclear export capacity, and CRM1 binding of the NP-NES3 consensus sequence mutants.
a<p>% virus titer ± SD compared with WT at the 46 h of replication kinetic assay from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g004" target="_blank">Fig. 4C</a>.</p>b<p>No viral rescue by reverse genetics.</p>c<p>% cell count ± SD with nuclear localization of NP from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g005" target="_blank">Fig. 5B</a>.</p>d<p>−, ±, + indicate not occur, partially occur, occur of nuclear export capacity, respectively derived from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g006" target="_blank">Fig. 6</a>.</p>e<p>% intensity of the pull-downed NP band compared with WT from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g007" target="_blank">Fig. 7C</a>.</p
Conservation of hydrophobic (Φ) residues within the NP-NES3 consensus sequences of influenza A viruses.
<p>Conservation of hydrophobic (Φ) residues within the NP-NES3 consensus sequences of influenza A viruses.</p
Production, protein expression, and replication kinetics of NES mutant viruses.
<p>(A) Wild-type NES or mutant viruses (M1-NES, NS1-NES, NS2-NES1, NS2-NES2, NP-NES1, NP-NES2, and NP-NES3) were produced via reverse genetics by co-culturing HEK-293T and MDCK cells transfected with eight viral genomic plasmids (pHH21 containing PA, PB1, PB2, HA, NA, NP, M, and NS) and four viral protein expression plasmids (pCAGGS containing PA, PB1, PB2, and NP) for 72 h. Supernatants containing viruses were collected, clarified, and titrated in a plaque assay on MDCK cells. Viral titration was performed in triplicate, and the plaque forming units PFU/ml (mean±SD) was calculated and plotted (upper panel). Viral protein expression in the producer cells was compared by collecting the cells, lysing them, and separating the proteins on 10% SDS-PAGE gels. Proteins were then blotted with either an anti-WSN antibody (Ab) or an anti-actin monoclonal antibody (MAb) (lower panel). The HA, NP, M1, and M2 proteins are shown alongside molecular weight markers. (B) Expression of NP-NES wild-type and mutant proteins was compared by transfecting NP/pHH21, NP/pCAGGS, PA/pCAGGS, PB1/pCAGGS, or PB2/pCAGGS into HEK-293T cells for 48 h. The cells were then lysed and blotted with either an anti-WSN Ab or an anti-actin MAb. (C) MDCK cells were infected with equal amounts of each of the viruses (MOI = 0.001) described in (A) and then cultured. At the indicated time-points post-infection, the viruses were collected and titrated in a plaque assay. Data are expressed as the mean (±SD) PFU/ml from triplicate titrations.</p