Anti-FLAG(F4045) and anti-HA(H9658) antibodies were purchased from Sigma-Aldrich(St. Louis, MO, USA). Goat anti-mouse secondary antibody(sc-2005), anti-β-actin antibody(sc-130656), and Protein A/G PLUS Agarose(sc-2003) were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA, USA). Cy3-labeled goat anti-rabbit IgG antibody(P0183) was purchased from Beyotime Institute of Biotechnology(Jiangsu, China). MG-132(M7449) was purchased from Sigma-Aldrich. Anti-Ub mouse monoclonal antibody, FK2(BML-PW0150-0025), for the detection of mono-and polyubiquitinylated conjugates, was purchased from Enzo Life Sciences, Inc.(Farmingdale, NY, USA).
pCAGGS-P6-HA and pEGFP-P6 plasmids were gifts from Dr. Stanley Perlman. The PCR primers employed in the synthesis of the constructs are shown in Table 1. For yeast two-hybrid screening, P6 and P6-N(40 aa) were PCR amplified and inserted into the EcoR I and Sal I restriction sites of the pGBKT7 vector. P6-C(24 aa) was PCR amplified and inserted into the Nde I and Sal I restriction sites of the pGBKT7 vector. For mammalian expression, pEF-FLAG-P6 was constructed by inserting PCR amplified P6 into the Xho I and EcoR I restriction sites of the pEF-FLAG vector. For P6 mutant(M1, M2, and M3) expression, the PCR products were digested with Xho I and EcoR I and cloned into the pEF-FLAG vector. The human Nmi sequence was amplified by PCR using cDNA from human embryonic kidney(HEK)293T cells as the template and was inserted into the SalI and NotI restriction sites of the pRK-HA vector or inserted into the BamH I and Sal I sites of the pRK-FLAG vector. Constructs were confirmed by sequencing.
Table 1. The primers used in the study
MatchmakerTM GAL4 Two-Hybrid System 3 was purchased from Clontech Laboratories, Inc.(Mountain View, CA, USA), which includes a human cDNA library, a yeast strain AH109, empty vectors pGBKT7, pGADT7 and control vectors pCL1, pGADT7-T and pGBKT7-53. Yeast two-hybrid screening and related assays were performed according to the manufacturer's instructions. For fluorescence imaging, post-transfection cells were incubated with polyclonal mouse anti-HA antibody for 2 h and Cy3-labeled goat anti-mouse IgG for 1 h and then observed using laser confocal microscopy(FV1000, Olympus, Tokyo, Japan).
Assays were performed as described previously(Chen et al., 2013) with the following minor modifications: for ubiquitination immunoprecipitation, cell extracts were denatured by boiling for 5 min with lysis buffer(1% Tritox X-100, 20 mmol/L Tris-HCl, pH 7.4, 150 mmol/LNaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L DTT and protease cocktail) containing 1% sodium dodecyl sulfate(SDS). Next, the denatured extracts were diluted with lysis buffer at 10× the extract volume and were immunoprecipitated with mouse anti-HA antibody. Protein ubiquitination was analyzed by western blotting with the anti-Ub antibody, FK2.
Total RNA was extracted from cultured 293T cells with TRIzol Reagent(Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Gene transcripts were semi-quantified by PCR and DNA electrophoresis, and normalized to the housekeeping gene GAPDH. Primers for RT-PCR of Nmi were: sense 5′-GCTCTAGAATGGCAACGGATGAAAAGGA-3′; anti-sense 5′-CAAGCTTCTATTCTTCAAAGTATACT ATGTGA-3′. The primers used for RT-PCR of GAPDH were: sense 5′-GACATCAAGAAGGTGGTGAA-3′; anti-sense 5′-TGTCATACCAGGAAATGAGC-3′.
Antibodies and chemical reagents
Yeast two-hybrid screening and fluorescence imaging
Immunoprecipitation and western blot analysis
As a highly conserved accessory protein in SARSrelated coronaviruses, P6 has attracted much interest from researchers. Previous studies have shown that P6 is involved not only in virus replication and infection but also in host gene regulation and immune responses(Spiegel et al., 2005; Kopecky-Bromberg et al., 2007; Kumar et al., 2007; Netland et al., 2007; Zhao et al., 2009). To further explore the function of P6 and identify its host-cell binding partners involved in SARS-CoV infection, we employed a yeast two-hybrid system to screen a human spleen cDNA library purchased from Clontech Laboratories, Inc. for proteins that interact with P6. Fourteen positive clones were characterized and further analyzed to eliminate repeated sequences. Subsequently, eight host proteins were identified and these are shown in Table 2.
Table 2. Potential host cell proteins inter racting with severe acute respiratory syndrome coronavirus (SARS-CoV) protein 6 (P6)
From these potential P6-interacting proteins, Nmi was selected for further study. Nmi has been previously shown to be induced by IFN and to regulate IFNdependent transcription(Zhu et al., 1999). Previous studies have evidenced that P6 can inhibit the gene expression induced by ISRE promoter after Sendai virus(SeV) infection(Kopecky-Bromberg et al., 2007), although the mechanism underlying this process remains unclear. We speculated that P6 might regulate IFN-dependent transcription through interactions with Nmi to inhibit Nmiregulated IFN signaling.
In our yeast two-hybrid experiments, we identified two positive clones of Nmi that interact with SARS-CoV P6(prey D and I, Figure 1A). Both contain the Nmi 1-260 aa region, suggesting that the interaction domain of the protein might be located in this region. We also cloned the hydrophobic N-terminal region(P6-N) and hydrophilic C-terminal tail(P6-C) of P6 with yeast two-hybrid BD vectors and tested their interacting properties with Nmi. The results showed that the 24 hydrophilic C-terminal residues of P6, but not the 40 hydrophobic N-terminal residues, could interact with Nmi(Figure 1B). Therefore, we concluded that the C-terminal hydrophilic tail of SARS-CoV P6 facilitates the protein's interaction with host Nmi protein.
Figure 1. Severe acute respiratory syndrome coronavirus (SARS-CoV) protein 6 (P6) interacts with N-myc (and STAT) interactor (Nmi) in yeast and in vivo. (A) Yeast strain AH109 was transformed with plasmids and plated onto synthetic minimal medium (SD) plates as indicated in A. Prey-I: AD-Nmi 1–260 aa. Prey-D: AD-Nmi 1-267 aa. Positive control: BD-p53 + AD-T. Negative control: BD-lam + AD-T. (B) Positive colonies were retested using the X-Gal assay. The blue color shows positive interaction. P6-N (40 aa): BD-P6 1–40 aa. P6-C (24 aa): BD-P6 40–63 aa. Positive controls: pCL1 and BD-p53 + AD-T. Negative control: BD-lam + AD-T. AD: activation domain, BD: binding domain.
To determine whether P6 also interacts with Nmi in vivo, we cloned P6 and Nmi into eukaryotic vectors and transfected these plasmids into 293T cells. The interaction between Nmi and P6 was detected by colocalization analysis using immunofluorescence. Using confocal microscopy, we observed that the expression of P6 and Nmi, shown by green and red fluorescence, respectively, was colocalized. This was represented by the merging of the fluorescence colors into yellow fluorescence at particular spots within cells(Figure 2A, 2B), indicating that P6 could interact with Nmi in vivo. In addition, although single transfection of P6 exhibited a similar pattern to co-expression, since green spots were observed in both instances(Figure 2A, 2B), P6 seemed to modify the subcellular localization of Nmi. In single transfection, Nmi was spread evenly throughout the cytoplasm(Figure 2B); while when co-expressed with P6, Nmi aggregated in some areas to colocalize with P6(Figure 2A). These results suggest that through their interaction, P6 could recruit Nmi.
Figure 2. Colocalization of severe acute respiratory syndrome coronavirus (SARS-CoV) protein 6 (P6)-GFP and N-myc (and STAT) interactor-hemagglutinin Nmi-HA in human embryonic kidney 293T cells. (A) 293T cells were transfected with 0.5 µg pEGFP-P6 and 0.5 µg pRK-Nmi-HA. 24 h post-transfection, cells were incubated with polyclonal mouse anti-HA antibody for 2 h and Cy3-labeled goat anti-mouse IgG for 1 h and observed under a fluorescence microscope. (B) 293T cells were transfected, fixed, and labeled as in (A). Cells were observed under a fluorescence microscope. GFP: green fluorescent protein.
FLAG-tagged Nmi, either alone or together with HAtagged P6, was(co) transfected into 293T cells and their respective protein levels were determined using western blot analysis. The results showed that P6 could promote Nmi protein degradation in a dose-dependent manner(Figure 3A). To further confirm that P6 regulates Nmi at the protein level, RT-PCR was adopted to show that P6 does not affect Nmi mRNA production(Figure 3B). These results indicated that P6 could regulate Nmi protein levels by promoting Nmi degradation. To examine whether P6-promoted Nmi degradation is dependent on the UPP, proteasome inhibitor MG-132 was used to treat the cells and Nmi ubiquitination was examined by HANmi immunoprecipitation followed by anti-Ub western blot analysis. The results showed that the P6-mediated Nmi degradation could be partially recovered by MG-132 treatment(Figure 3C, 3D), and that Nmi ubiquitination was increased from control levels by P6 cotransfection and was further increased by MG-132 treatment(Figure 4A). These results indicated that P6 could stimulate Nmi ubiquitination and thus promote Nmi degradation through the proteolytic UPP.
Figure 3. Severe acute respiratory syndrome coronavirus (SARS-CoV) protein 6 (P6) mediates ubiquitin-dependent proteosomal degradation of N-myc (and STAT) interactor (Nmi). (A) Western blotting (WB), with indicated antibody, for human embryonic kidney 293T cells transfected with 2 µg pRK-Nmi-FLAG together with increasing amounts (0.2 µg, 0.5 µg, 1 µg, 2 µg) of pCAGGS-P6-HA. Cell extracts were analyzed 24 h post-transfection. (B) Reverse transcription-PCR results for 293T cells transfected with 2 µg pRK-Nmi-FLAG together with 2 µg pCAGGS-P6-HA, pEGFP-P6, or their respective empty vectors pCAGGS and pEGFP. Total RNA was extracted 48 h posttransfection. (C) Immunoblots, with the indicated antibodies, of 293T cells transfected with 2 µg pCAGGS-P6-HA and 2 µg pRK-Nmi-FLAG and treated with dimethyl sulfoxide (DMSO)/20 µmol/L MG-132 for 4 h before harvesting of cell extracts. (D) The relative expression of Nmi (Figure 3C, first panel, lane 1, 4, and 5) was calculated and graphed with grey intensity analysis. The bar graphs indicate mean values ± SD of triplicate samples from three independent experiments. * indicates P < 0.05.
Figure 4. The activity N-myc (and STAT) interactor (Nmi) degradation-mediating activity of severe acute respiratory syndrome coronavirus (SARS-CoV) protein 6 (P6) mutants. Human embryonic kidney 293T cells were transfected as indicated, and treated with dimethyl sulfoxide (DMSO)/20 µmol/L MG-132 for 4 h. Cell extracts were harvested and analyzed by immunoprecipitation and western blotting with the antibodies indicated. (A) 6 µg pRK-Nmi-HA and 4 µg pEF-FLAG-P6 were transfected into 293T cells. (B) Diagram of P6 mutants. (C) 6 µg pRK-Nmi-HA was transfected with 4 µg pEF-FLAG-P6 wild type (WT) or M1, M2, or M3 mutant. (D) The relative expression of Nmi (cells in Fig. 4C, first panel) was calculated and graphed with grey intensity analysis. The bars indicate mean value ± SD of triplicate samples from three independent experiments. * indicates P < 0.05.
Based on our results of the yeast two-hybrid system and structural analysis, we predicted that the C-terminal of P6 might play the key role in its inhibitory activity on Nmi protein. Three P6 mutants with alanine mutations on amino acids 49 to 53, 54 to 58, and 59 to 63 were constructed and designated the names M1, M2, and M3(Figure 4B). Their capacity to induce Nmi degradation and ubiquitination was examined and compared with wild type(WT) P6. The results showed that M1 demonstrated similar activity to WT P6; enhanced Nmi ubiquitination and degradation. However, both M2 and M3 had no effect on Nmi ubiquitination and degradation(Figure 4C, 4D). These results further confirmed our conclusion that SARS-CoV P6 functions to promote Nmi ubiquitination and degradation through interacting with Nmi. In addition, we found that the C-terminal 54 to 63 aa of P6 are critical for its functions.