Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

Abstract

Porcine epidemic diarrhea virus (PEDV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleolus. The mechanism of nuclear translocation, and whether N protein associates with particular nucleolar components, is unknown. In this study, we confirm that a nucleolar phosphoprotein nucleophosmin (NPM1) interacts and co-localizes with the N protein in the nucleolus. In vitro binding studies indicated that aa 148-294 of N and aa 118-188 of NPM1 were required for binding. Interestingly, N protein importation into the nucleolus is independent of the ability of NPM1 to shuttle between the nucleus and the cytoplasm. Furthermore, overexpression of NPM1 promoted PEDV growth, while knockdown of NPM1 suppressed PEDV growth. In addition, binding of N protein to NPM1 protects it from proteolytic degradation by caspase-3, leading to increased cell survival. Taken together, our studies demonstrate a specific interaction of the N protein with the host cell protein NPM1 in the nucleolus. The results suggest potential linkages among viral strategies for the regulation of cell survival activities, possibly through an interaction of N protein with NPM1 which prevents its proteolytic cleavage and enhances cell survival, thus ultimately promoting the replication of PEDV.

Figure 1. Western blot analysis of N protein in nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells at 48 h.

Western blot analysis of nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells with an anti-GAPDH mAb (A), anti-PCNA mAb (B), and anti-N mAb (C). (A,B and C) Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells. (C) Moc, mock-infected cells; Inf, PEDV-infected cells. The arrowheads indicate purified bands that are the same sizes as GAPDH (A), PCNA (B), and N (C) proteins. (D) Western blot analysis of N protein in nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells at different times with an anti-GAPDH mAb, anti-PCNA mAb, and anti-N mAb. Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells.

Figure 2. Interaction of PEDV N with NPM1.

Co-IP of NPM1 with N protein. HEK293T cells were co-transfected with the indicated plasmids (+) or empty vectors (−) and the whole-cell lysates (WCL) obtained at 48 hpt were immunoprecipitated with anti-Myc (A) or anti-Flag (B) mAb. After separation by SDS-PAGE, proteins were detected by immunoblotting with the indicated antibodies. A 5% aliquot of WCL was also probed to confirm protein expression. The identities of the protein bands are indicated on the right. (C) Co-IP of PEDV N protein with endogenous NPM1. PEDV-infected (+) or mock-infected (−) Vero E6 cells were used for IP with anti-NPM1 protein mAb and immunoblotted with the indicated antibodies. The identities of the bands are shown on the right. (D) GST-pull down assay. Glutathione beads conjugated to GST or the GST-NPM1 fusion protein were incubated with recombinant Myc-N. After washing, proteins were eluted from the beads and SDS-PAGE was performed. The presence of N protein was detected by immunoblotting with anti-Myc mAb. GST and GST-NPM1 protein expression was confirmed by immunoblotting with mouse anti-GST mAb. (E) Co-localization of N protein with NPM1. Vero E6 cells were co-transfected with pAcGFP-N and pDsRed-NPM1. The PEDV N protein is colored green and the NPM1 fusion protein colored red. Merged images are also presented, and the position of the nucleus is indicated by DAPI (blue) staining in the merged images. The nucleolus (No) is arrowed where appropriate. Lower panels show boxed regions at high magnification. (F) Co-IP of HEK293T cells co-transfected with recombinant constructs encoding Myc-N and 3×Flag-tagged porcine NPM1.

Figure 3. GST-pull down mapping of binding domains between N protein and NPM1.

(A,B,C and D) GST-pull down mapping domains of N protein for NPM1 binding. (A) Schematic representation of full-length and deletion mutants of GFP-tagged N protein. (B,C and D) Glutathione beads conjugated to the GST-NPM1 fusion protein were incubated with full-length and deletion mutants of GFP-tagged N protein and the pulled down proteins were immunoblotted against GFP. (E and F) GST-pull down probing regions of NPM1 for N protein binding. (E) Schematic diagram of full-length and deletion mutants of GST-tagged NPM1. (F) GST-pull down assays were performed by incubation of purified GST-NPM1 or its deletion mutants with Myc-N protein. Pull down fractions were detected by immunoblotting against Myc mAb.

Figure 4. NPM1 phosphorylation or sumoylation has no effect in mediating its binding to PEDV N protein.

HEK293T cells were co-transfected with the indicated plasmids, and the WCL obtained at 48 hpt were immunoprecipitated with anti-Flag mAb. After separation by SDS-PAGE, proteins were detected by immunoblotting with the indicated antibodies. A 5% aliquot of WCL was also probed to confirm protein expression. The identities of the protein bands are indicated on the right. Densitometric data for Myc-N/3×Flag-NPM1 and mutants from three independent experiments are expressed as mean ± SD.

Figure 5. N protein is imported into the nucleolus independently of NPM1.

(A) The NPM1 fusion protein as a marker of the nucleolus is colored red. The nucleolar localization of N in transfected cells was clearly observed at 42–42.5 hpt. As shown, a small amount of N was observed in the nucleolus at 42 hpt (t = 30 min). N protein accumulated continuously in the nucleolus of transfected cells until t = 60 min and was exported from the nucleolus at t = 61–65 min. This figure shows snapshots of the cells from the time-lapse movie (Video S3 in the supplemental materials). Data are representative of one of three independent experiments. Real-time visualization of the kinetics of the nucleolar localization of N protein indicated that the process was rapid, taking only 30 min in total. (B) Knockdown of NPM1 protein levels following siRNA treatment. Vero E6 cells transfected with no siRNA (Mock), scrambled siRNA (siScr), left untreated (No treat) or with different concentrations (mM) of siRNAs targeting NPM1 (siNPM1) (as indicated at the top of each lane) were harvested 48 hpt. Endogenous NPM1 protein levels were detected by immunoblotting using antibodies directed against the indicated proteins. (C and D) Western blot analysis of Myc-N protein in nuclear and cytoplasmic fractions of NPM1-knockdown cells (C) or Ectopic NPM1-overexpression cells (D) at 48 hpt with anti-GAPDH mAb, anti-PCNA mAb, anti-NPM1 mAb, anti-Myc mAb and anti-Flag mAb. Nuc, nuclear fraction; Cyt, cytoplasmic fraction; cell, whole cells. Densitometric data for Nuc/Cell (Myc-N) from three independent experiments are expressed as mean ± SD.

Figure 6. Overexpression of NPM1 promotes N protein expression and PEDV growth.

(A and B) Ectopic expression of NPM1 in Vero E6 cells. Vero E6 cells were transfected with p3×Flag-NPM1 (+) or empty vector (–) for 12 h, followed by infection with PEDV at a MOI of 0.1 for 48 h (A) or 60 h (B). The levels of expression of 3×Flag-NPM1, N, or β-actin (loading control) (A and B) proteins in cell lysates were analyzed by western blot of cell lysates at the indicated time points after infection. Densitometric data for N/actin from three independent experiments are expressed as mean ± SD. (C) Promotion of PEDV growth in NPM1-overexpressing cells. Transfection and infection conditions were as described for panel A and B, and the titers of virus in the supernatants collected at 48 and 60 hpi were determined by the Reed–Muench method. Error bars represent the standard errors of the means from three independent experiments. P values are indicated on the bars. (D and E) Expression of endogenous NPM1 protein in Vero E6 cells infected with PEDV at various time points. Cells were infected with PEDV at MOI of 0.1 for 48 h or 60 h. The levels of expression of N, NPM1, or β-actin (loading control) (D and E) proteins in cell lysates were analyzed by western blot of cell lysates at the indicated time points after infection. Densitometric data for NPM1/actin from three independent experiments are expressed as mean ± SD.

Figure 7. Knockdown of NPM1 inhibits PEDV growth and N protein expression.

(A and B) PEDV N protein is reduced in NPM1-knockdown cells. Vero E6 cells treated with 200 nM siNPM1, No treat, Mock or siScr for 48 h were infected with PEDV for 48 h (A) or 60 h (B). Endogenous NPM1 was detected by western blot with anti-NPM1 mAb and PEDV N protein was detected by western blot with anti-N protein mAb. Densitometric data for N/actin and NPM1/actin from three independent experiments are expressed as mean ± SD. (C) PEDV titers in NPM1-knockdown cells. siRNA transfection and virus infection were performed as in panel A and B, and the virus titers in supernatants collected at 48 and 60 hpi were determined by the Reed–Muench method. Error bars represent the standard error of the mean from three independent experiments. P values are indicated above the bars.

Figure 8. N protein binding prevents NPM1 proteolytic cleavage and enhances cell survival.

(A and B) N protein binding prevents NPM1 proteolytic cleavage. Vero E6 cells were transfected with pMyc-N or empty vector for 24 h and then treated with or without 100 μM of Ac-DEVD-CHO (caspase-3 inhibitor) for 6 h. The cells were treated with or without 250 nm of STS for 18 h. The western blots were probed for freshly extracted proteins with antibodies against NPM1, Myc, GAPDH and caspase-3. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. CF, cleavage fragment. (C) N protein enhances the antiapoptotic effect of NPM1. Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h. Genomic DNA was loaded on to a 2% agarose gel. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. (D) Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h, then TUNEL and DAPI staining to examine the apoptotic cell death. Statistical results represent means ± SD of apoptotic cell counts from six different fields (right).