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

Abstract

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.

Keywords: CRM1; influenza A virus; nuclear export; nucleoprotein; phosphorylation; ubiquitination.

Figure 1

NP Y78 is a conserved phosphorylation site. (A) The Phos-tag SDS-PAGE gel of phosphorylated NP immunoprecipitated with rabbit anti-NP antibody. The IAV (WSN)-infected 293 T cells were lysed in lysis buffer supplemented with complete protease inhibitor cocktail and a phosphatase inhibitor phosSTOP. The extra band in untreated cells was considered to be a phosphorylated band compared to alkaline phosphatase (ALP)-treated cells. The band was digested and subjected to LC-MS/MS analysis. The locations of NP and phosphorylated NP bands (^pNP) are indicated by arrows. (B) LC-MS/MS analysis of the phosphorylated band. The band was identified as the IAV NP. The identified polypeptide sequence is indicated in blue, and the phosphorylated tyrosine site is indicated in red. (C) Detection of tyrosine-phosphorylated NP. A549 cells infected with viruses (WT or Y78F WSN, MOI = 1) were lysed at 12 h.p.i. and then were incubated with NP antibody and protein G agarose beads. The uninfected cells immunoprecipitated with anti-NP antibody and the infected cells immunoprecipitated with non-specific IgG served as controls. The immunoprecipitated NPs of the WT and Y78F WSN viruses were detected using anti-NP antibody (αNP) or anti-p-Tyr antibody (α^pY) (top). The relative density of phosphorylated NP was normalized to total NP (below). Data are shown as mean + SD (n = 3). Difference between WT and Y78F mutant viruses was tested using unpaired Student’s t-test. ^**p < 0.01. (D) Schematic diagram of NP functional domains, including Y78, nuclear localization sequence (NLS), nuclear export sequence (NES), nuclear aggregation sequence (NAS), basic loop and tail loop region.

Figure 2

NP Y78 phosphorylation affects IAV replication and polymerase activity. (A) Rescue of WT and Y78 mutant WSN viruses using the 12-plasmid reverse genetic system. The supernatants of 293 T cells were harvested at 72 h.p.t. and used for plaque assays in MDCK cells. N.D represents failed rescue; SD represents standard deviation. (B) Multi-cycle growth curves of viruses. A549 cells were respectively infected with WT and Y78F mutant WSN viruses (MOI = 0.001). Then the supernatants of virus-infected cells were examined by plaques assays at different time points. Data are shown as mean + SD (n = 3). Difference between WT and Y78F mutant viruses was tested using unpaired Student’s t-test. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. (C) The expression levels of M1 RNAs (mRNA, cRNA, and vRNA) were tested by real-time PCR. 293 T cells were transfected with the 12-plasmid reverse genetic system containing NP WT, Y78E, Y78F, or empty vector (Neg) plasmid for 48 h. Data are shown as mean + SD (n = 3). Differences between mutant and WT NPs were evaluated using one-way analysis of variance (ANOVA) followed by Dunnett’s test. ^**p < 0.01; ^***p < 0.001. (D) The polymerase activity of WT and mutant NPs were determined by luciferase assays. Luciferase activity was measured at 30 h after transfection of viral proteins (PB1, PB2, and PA); β-gal; and the cNS-Luc plasmid in 293 T cells. Differences of luciferase activities between the mutant and WT NPs were evaluated using one-way ANOVA followed by Dunnett’s test (below). Data are shown as mean + SD (n = 3). ^**p < 0.01; ^***p < 0.001, and the expressions of NPs were analyzed by western blotting (top).

Figure 3

Y78 phosphorylation inhibits the nuclear export of NP. (A) The cellular localizations of WT and mutant NPs (Y78F and Y78E) were determined using IFAs. 293 T cells were transfected with plasmids expressing WT or mutant NPs. Cells were fixed at 12, 24, and 36 h.p.t. The nucleus was stained with DAPI (blue), and the subcellular distribution of NP (red) was analyzed (top). At least 600 cells from each condition were scored as predominantly nuclear (N), nuclear and cytoplasmic (N + C), or predominantly cytoplasmic (C) (below), and the percentage of cells representing each is shown. Differences of cellular localization between the mutant and WT NPs were evaluated using one-way ANOVA followed by Dunnett’s test. Data are shown as mean + SD (n = 3). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. (B) Immunoblot analysis of the subcellular location of NP. 293 T cells were transfected with plasmid expressing WT, Y78F, or Y78E NP. The nuclear and cytoplasmic fractions were separated at 24 and 36 h.p.t. and detected using anti-NP and anti-Lamin B1 or anti-Hsp70 antibody (left). Nuclear NP was normalized to Lamin B1, and cytoplasmic NP was normalized to Hsp70. Data are shown as mean + SD (n = 3). The differences of N/C ratio between mutant and WT NPs were evaluated using one-way ANOVA followed by Dunnett’s test (right). ^**p < 0.01; ^***p < 0.001.

Figure 4

Y78 phosphorylation negatively regulates the binding of NP to CRM1. (A) Co-immunoprecipitation (CO-IP) experiments of WT and mutant NPs with CRM1. The NP (WT, Y78F, and Y78E) with pcDNA4.0/TO-tagged plasmid and pCDNA3.0-FLAG-CRM1 plasmid were co-transfected into 293 T cells, and cells were lysed at 24 h.p.t. FLAG-CRM1 was immunoprecipitated with anti-FLAG agarose, and the associated NP was detected using rabbit anti-NP antibody (left). The relative density of immunoprecipitated NPs was normalized to immunoprecipitated CRM1 (right). Data are shown as mean + SD (n = 3). Differences between mutant and WT NPs were evaluated using one-way ANOVA followed by Dunnett’s test. ^***p < 0.001. (B) The co-localizations of CRM1 with NPs were examined by IFAs. The 293 T cells expressing FLAG-CRM1 and pCDNA4.0-NP (WT, Y78F, and Y78E) were fixed and stained with anti-NP (red) and anti-FLAG (green) antibodies at 24 h.p.t. The nucleus was stained with DAPI (blue).

Figure 5

Y78 phosphorylation status regulates the nuclear export of NEP-M1-vRNP complex. The co-localizations of NP with M1/NEP/PB1 were examined by IFAs. The 12-plasmid reverse genetic system containing WT, Y78F, or Y78E NP plasmid was transfected into 293 T cells. At 12 and 36 h.p.t., cells were fixed and stained with anti-NP (red) and anti-M1/NEP/PB1 antibodies (green). The nucleus was stained with DAPI (blue).

Figure 6

Janus and Src kinases are involved in Y78 phosphorylation-regulated NP nuclear export. (A) Effect of tyrosine kinase inhibitors on NP phosphorylation. A549 cells were infected with WSN or Y78F mutant virus (MOI = 1) for 12 h and then treated with the tyrosine kinase inhibitors Imatinib (10 μM), Dasatinib (10 μM), and AG490 (50 μM) for 6 h, with DMSO as a control (the inhibitors were dissolved in DMSO). Then cells were lysed and incubated with NP antibody and protein G agarose beads. The uninfected cells immunoprecipitated anti-NP antibody and the infected cells that were treated with DMSO and immunoprecipitated with non-specific IgG served as controls. The immunoprecipitated NPs were detected using anti-NP antibody (αNP) or anti-p-Tyr antibody (α^pY). (B) Effect of tyrosine kinase inhibitors on the nuclear export of NP. A549 cells were infected with WSN (MOI = 0.1) and treated with CHX (100 μg/ml) and several tyrosine kinase inhibitors, including Imatinib (10 μM), Dasatinib (10 μM) and AG490 (50 μM) for 2 h at 10 h.p.i., with DMSO as a control. Cells were fixed and stained with anti-NP (red) antibody. The nucleus was stained with DAPI (blue). At the same time, some cells only infected with WSN were fixed at 6 h.p.i. (left). At least 600 cells from each group were scored as N, N + C, or C, and the percentage of cells representing each is shown. Data are shown as mean + SD (n = 3). Differences of cellular localization between the tyrosine kinase inhibitor- and DMSO-treated groups were evaluated using one-way ANOVA followed by Dunnett’s test (right). ^**p < 0.01; ^***p < 0.001. (C) Effects of tyrosine kinase inhibitors on the binding of NP to CRM1. 293 T cells were co-transfected FLAG-CRM1 and MYC-NP plasmids, then tyrosine kinase inhibitors Imatinib (10 μM), Dasatinib (10 μM), or AG490 (50 μM) were added to medium at 24 h.p.t., with DMSO as a control. Cells were lysed for CO-IP assays after 6 h post inhibitor treatment (left). The relative density of immunoprecipitated NPs was normalized to immunoprecipitated CRM1 (right). Data are shown as mean + SD (n = 3). Differences of NP-CRIM1 interaction between the tyrosine kinase inhibitor- and DMSO-treated groups were evaluated using one-way ANOVA followed by Dunnett’s test. ^*p < 0.05; ^**p < 0.01.

Figure 7

Y78 phosphorylation inhibits NP ubiquitination. (A) Effect of Y78 phosphorylation on NP ubiquitination. 293 T cells were transfected with FLAG-tagged NPs and HA-tagged ubiquitin (HA-Ub). Cell extracts were immunoprecipitated with anti-FLAG beads at 30 h.p.t., and the ubiquitination was detected using rabbit anti-HA antibody. (B) Effect of tyrosine kinase inhibitors on NP ubiquitination. 293 T cells were transfected with FLAG-tagged WT NP and HA-tagged ubiquitin (HA-Ub). At 30 h.t.p., cells were treated with DMSO or inhibitors for 6 h. Cell extracts were immunoprecipitated with anti-FLAG beads and the ubiquitination was detected using rabbit anti-HA antibody.

Figure 8

Y78 phosphorylation regulates TRIM22-mediated NP ubiquitination. (A) Effect of TRIM22 and CNOT4 on the ubiquitination of NPs. Immunoblot analysis of lysates in 293 T cells transfected with various combinations of plasmids for 30 h, followed by immunoprecipitation with anti-FLAG beads. (B) Effect of TRIM22 on the stability of IAV NPs. 293 T cells were transfected with FLAG-NP (WT, Y78F, or Y78E) plasmid, along with MYC-TRIM22 or an empty vector for 30 h. Cells were lysed and then detected with corresponding antibodies (top). The relative density of NPs was normalized to β-actin (below). Data are shown as mean + SD (n = 3). Differences of NP stability between the TRIM22- and Vector-transfected groups were tested using unpaired Student’s t-test. ^**p < 0.01. (C) Effect of Y78 phosphorylation on the interaction of TRIM22 with WT or mutant NPs. 293 T cells were transfected with MYC-NP (WT, Y78F, and Y78E) and FLAG-TRIM22 and lysed at 30 h.p.t. FLAG-TRIM22 was immunoprecipitated with anti-FLAG beads, and the associated NPs were detected using rabbit anti-NP antibody (top). The relative density of immunoprecipitated NPs was normalized to immunoprecipitated TRIM22 (below). Data are shown as mean + SD (n = 3). Differences between mutant and WT NPs were evaluated using one-way ANOVA followed by Dunnett’s test. ^**p < 0.01; ^***p < 0.001.