Molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus

Abstract

The nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. The porcine epidemic diarrhea virus (PEDV), coronavirus nucleocapsid (N) protein, plays important roles in the process of virus replication and cellular infection. Virus infection and transfection showed that N protein was predominately localized in the cytoplasm, but also found in the nucleolus in Vero E6 cells. Furthermore, by utilizing fusion proteins with green fluorescent protein (GFP), deletion mutations or site-directed mutagenesis of PEDV N protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), 71-90 in region 1 of the N protein was sufficient for nucleolar localization and R87 and R89 were critical for its function. We also identified two nuclear export signals (NES, aa221-236, and 325-364), however, only the nuclear export signal (aa325-364) was found to be functional in the context of the full-length N protein. Finally, the activity of this nuclear export signal (NES) was inhibited by the antibiotic Lepomycin B, suggesting that N is exported by a chromosome region maintenance 1-related export pathway.

Figure 1

(A) Characterization of the mouse anti-N polyclonal antibody. Lane 1, negative control; Lane 2, N protein from PEDV CV777; M, protein marker; (B) Analysis of N protein in transiently transfected Vero E6 cells. M, protein marker; Lane 1, pcDNA3.1 (+) control; Lane 2, N protein.

Figure 2

(A) Detection of PEDV N protein by indirect immunofluorescence in cells infected with PEDV CV777; (B) Detection of PEDV N protein by indirect immunofluorescence in cells transfected with pcDNA3.1 (+)-N. After 24 h, infected or transfected, cells were fixed and analyzed by indirect immunofluorescence using mouse anti-N polyclonal antibody (Green) and stained with PI (red) to visualize the nuclear DNA. Differentially fluorescing images were gathered separately using confocal microscopy. Images were obtained with a 63× oil objective.

Figure 3

Live cell and confocal microscopy of the subcellular localization of fluorescent fusion proteins: AcGFP, AcGFP-N, AcGFP-NR1, AcGFP-NR2, AcGFP-NR3, AcGFP-NR1+2, and AcGFP-NR2+3 proteins. Vero cells were visualized 24 h post-transfection in culture conditions. Confocal analysis of the subcellular localization of AcGFP, AcGFP-N, AcGFP-NR1, AcGFP-NR2, AcGFP-NR3, AcGFP-NR1+2, and AcGFP-NR2+3 proteins in cells co-expressing B23.1-DsRed, at 24 h post-transfection. The PEDV fusion peptides are colored green and the B23.1 fusion protein colored red. Merged images are also presented. The nucleolus (No) is arrowed where appropriate.

Figure 4

Live cell and confocal microscopy of the sub-cellular localization of fluorescent fusion proteins: AcGFP-NR1_1–50, AcGFP-N1_51–100, AcGFP-N1_100–147. The PEDV fusion peptides are colored green and the B23.1 fusion protein colored red. Merged images are also presented. The nucleolus (No) is arrowed where appropriate.

Figure 5

Live cell and confocal microscopy of the sub-cellular localization of fluorescent fusion proteins: AcGFP-NR1_51–70, AcGFP-NR1_61–80, AcGFP-NR1_71–90, AcGFP-NR1_81–100. The PEDV fusion peptides are colored green and the B23.1 fusion protein colored red. Merged images are also presented. The nucleolus (No) is arrowed where appropriate.

Figure 6

Clustal W analysis of the PEDV N protein NoLS with known cellular and viral NoLSs. Red squares indicate the amino acids of conservation. The cellular and viral NoLSs are described in NoLS IBV N protein [34], NoLS PRRSV N [20], NoLS HTLV-1 Rex [35], NoLS HSV gamma1 34.5 [36], NoLS MDM2 [37], NoLS MDV MEQ [38], NoLS NF-kappa [39], NoLS Nuclear VCP-like protein (NVL2) [40], NoLS p 120 [41], NoLS surviving-deltaEx3 [42], NoLS (GGNNV) protein alpha [43], BHV-1 BICP27 [44], Earning-associated protein 1–19 [45], HSV-1 ICP27 [46], NoLS angiogen [47], NoLS Fibroblast growth factor-2 [48], NoLS herpes-mareks MEQ [38], NoLS HIC p40 [49], NoLS HIV-1 rev [50], NoLS HIV-1 Tat [51].

Figure 7

Confocal microscopy of the sub-cellular localization of fluorescent fusion proteins: AcGFP-NR2_148–220, AcGFP-NR2_221–294 AcGFP-NR2_221–240, AcGFP-NR2_241–260, and AcGFP-NR2_261–294. Fusion proteins are colored green and nucleus colored blue. Merged images are also presented. The nucleolus (No) is arrowed where appropriate.

Figure 8

Confocal microscopy of the sub-cellular localization of fluorescent fusion proteins: AcGFP-NR2_221DLVA-AAAA, AcGFP-NR2_225AVKD-AAAA, AcGFP-NR2_229ALKS-AAAA, AcGFP-NR2_233LGIG-AAAA and AcGFP-NR2_237ENPD-AAAA Fusion proteins are colored green and nucleus colored blue. Merged images are also presented. The nucleolus (No) is arrowed where appropriate.

Figure 9

Confocal microscopy of the sub-cellular localization of fluorescent fusion proteins AcGFP-N_∆221–236. Fusion proteins are colored green and nucleus colored blue. Merged images are also presented. The nucleolus (No) is arrowed where appropriate.

Figure 10

Confocal microscopy of the sub-cellular localization of fluorescent fusion proteins: AcGFP-N_295–394 and AcGFP-N_345–441 AcGFP-N_295–324, AcGFP-N_325–364, AcGFP-N_365–394. Fusion proteins are colored green and nucleus colored blue. Merged images are also presented. The nucleolus (No) is arrowed where appropriate.

Figure 11

The nuclear export mechanism of PEDV N. Vero E6 cells were transiently transfected with plasmids encoding pAcGFP-N, pAcGFP-NR3, pAcGFPNR3_365-394, with or without treatment with LMB, and examined live 24 h after transfection by confocal microscopy. Each image is representative of the majority of the cells observed in the same cells. The nucleolus (No) is arrowed where appropriate.