Subcellular localization and membrane association of SARS-CoV 3a protein

Abstract

SARS-CoV 3a protein was a unique protein of SARS coronavirus (SARS-CoV), which was identified in SARS-CoV infected cells and SARS patients' specimen. Recent studies revealed that 3a could interact specifically with many SARS-CoV structural proteins, such as M, E and S protein. Expressed 3a protein was reported to localize to Golgi complex in SARS-CoV infected cells. In this study, it was shown that 3a protein was mainly located in Golgi apparatus with different tags at N- or C-terminus. The localization pattern was similar in different transfected cells. With the assay of truncated 3a protein, it was shown that 3a might contain three transmembrane regions, and the second or third region was properly responsible for Golgi localization. By ultra-centrifugation experiment with different extraction buffers, it was confirmed that 3a was an integral membrane protein and embedded in the phospholipid bilayer. Immunofluorescence assay indicated that 3a was co-localized with M protein in Golgi complex in co-transfected cells. These results provide a new insight for further study of the 3a protein on the pathogenesis of SARS-CoV.

Fig. 1

Expression and cellular localization of 3a within transfected cells. (A) Construction of 3a/pEGFP-N1, 3a/pEGFP-C1 and 3a/pCMV-myc. 3a from the SARS-CoV genome was cloned as a C-terminal fusion to the pEGFP-N1 vector, and as N-terminal fusion to the pEGFP-C1 and pCMV-myc vectors. (B) Western blotting analysis. 293 cells were transiently transfected with pEGFP-N1, 3a/pEGFP-N1, pEGFP-C1, 3a/pEGFP-C1, pCMV-myc and 3a/pCMV-myc plasmids separately. Cell lysates were prepared at 48 h after transfection and separated by SDS-PAGE. Proteins transferred to PVDF membrane was probed with monoclonal anti-GFP or anti-myc antibody. Sizes (kDa) of molecular mass markers were indicated on the left. (C) Cellular localization of 3a protein with different tags. 293 cells were transfected with described plasmids separately. Cellular localization of 3a was observed by scanning fluorescence confocal microscopy. Green represented EGFP fluorescence (1, 4 and 7) from original EGFP, 3a-EGFP or EGFP-3a fusion protein; blue (2, 5, 8 and 11) represented Hoechest stained cell nuclei; 3, 6, 9 and 12 images represented overlapping green and blue fluorescence. In panels 10, cells were labeled with FITC conjugated goat anti-mouse antibody to show the expression of myc-3a. All three panels of a row had the same field of view. (D) Cellular localization of 3a protein in different transfected cells. COS-7, Vero and A549 cells were transiently transfected with 3a/pEGFP-N1 separately. Cellular localization of 3a was observed at 24 h after transfection. Green represented EGFP fluorescence from expressed 3a-EGFP protein in different cells; red represented propidium idodine (PI) stained cell nuclei; yellow (in the overlay image) represented overlapping green and red fluorescence. All three panels had the same field of view. (E) Cellular localization of 3a protein at different time after transfection. 3a/pEGFP-N1 was transfected into 293 cells. At 12 h, 24 h, 36 h and 48 h after transfection, the cellular localization of 3a was viewed with fluorescence confocal microscopy separately. Green represented EGFP fluorescence from expressed 3a-EGFP protein.

Fig. 1

Expression and cellular localization of 3a within transfected cells. (A) Construction of 3a/pEGFP-N1, 3a/pEGFP-C1 and 3a/pCMV-myc. 3a from the SARS-CoV genome was cloned as a C-terminal fusion to the pEGFP-N1 vector, and as N-terminal fusion to the pEGFP-C1 and pCMV-myc vectors. (B) Western blotting analysis. 293 cells were transiently transfected with pEGFP-N1, 3a/pEGFP-N1, pEGFP-C1, 3a/pEGFP-C1, pCMV-myc and 3a/pCMV-myc plasmids separately. Cell lysates were prepared at 48 h after transfection and separated by SDS-PAGE. Proteins transferred to PVDF membrane was probed with monoclonal anti-GFP or anti-myc antibody. Sizes (kDa) of molecular mass markers were indicated on the left. (C) Cellular localization of 3a protein with different tags. 293 cells were transfected with described plasmids separately. Cellular localization of 3a was observed by scanning fluorescence confocal microscopy. Green represented EGFP fluorescence (1, 4 and 7) from original EGFP, 3a-EGFP or EGFP-3a fusion protein; blue (2, 5, 8 and 11) represented Hoechest stained cell nuclei; 3, 6, 9 and 12 images represented overlapping green and blue fluorescence. In panels 10, cells were labeled with FITC conjugated goat anti-mouse antibody to show the expression of myc-3a. All three panels of a row had the same field of view. (D) Cellular localization of 3a protein in different transfected cells. COS-7, Vero and A549 cells were transiently transfected with 3a/pEGFP-N1 separately. Cellular localization of 3a was observed at 24 h after transfection. Green represented EGFP fluorescence from expressed 3a-EGFP protein in different cells; red represented propidium idodine (PI) stained cell nuclei; yellow (in the overlay image) represented overlapping green and red fluorescence. All three panels had the same field of view. (E) Cellular localization of 3a protein at different time after transfection. 3a/pEGFP-N1 was transfected into 293 cells. At 12 h, 24 h, 36 h and 48 h after transfection, the cellular localization of 3a was viewed with fluorescence confocal microscopy separately. Green represented EGFP fluorescence from expressed 3a-EGFP protein.

Fig. 2

Subcellular localization of 3a protein. (A) Subcellular localization of 3a protein with fluorescence marker. 293 cells transfected with the 3a/pEGFP-N1 plasmid were grown for 24 h, washed with PBS, and stained with Golgi or ER fluorescence marker. Subsequent, cells were fixed, and then nuclear was stained with Hoechest or PI. 3a-EGFP protein (green, 1 and 5), Golgi fluorescence marker BODIPY TR C₅-ceramide (red, 2), ER fluorescence marker ER-Tracker™ Blue–White DPX (blue, 6) and merge image (4 and 8) were indicated. (B) Subcellular localization of 3a protein with Golgi-DsRed protein. 293 cells co-transfected with the 3a/pEGFP-N1 and Golgi/pDsRed-N1 plasmids. At 24 h after transfection, cells were fixed, and nuclear was stained with Hoechest. 3a-EGFP protein (green, 1), Golgi-DsRed marker (red, 2), DNA staining (blue, 3) and merge image (4) were indicated.

Fig. 3

Expression and cellular localization of truncated 3a protein. (A) DNA constructs of deletant 3a protein. The truncated SARS-CoV 3a proteins were cloned into pEGFP-N1 vector as C-terminal fusion to EGFP. The different mutants were constructed based on bioinformative studies of SARS-CoV 3a. Kozak consensus translation initiation site was indicated. The animo acid positions for 3a were given at the low (a, b and c represented the three transmembrane regions). C or N from localization represented the cytoplasm or nuclear localization. (B) Western blotting analysis. 293 cells were transiently transfected with described plasmids separately. Cell lysates were prepared at 48 h post-transfection and separated by SDS-PAGE. Proteins transferred to PVDF membrane was probed with anti-GFP antibody. Sizes (kDa) of molecular mass markers were indicated on the left. (C) Cellular localization of truncated 3a protein. At 24 h post-transfection with indicated mutants, 293 cells were analyzed by confocal fluorescence microscopy. Left row (green) showed fusion EGFP fluorescence; middle row (red) showed PI stained cell nuclei; right row (yellow) represented overlapping green and red fluorescence. The fluorescent pattern of 3a D1–147, D1–103 and D77–274 (A–F, V–X), like wild-type 3a, was distributed in the cytoplasm and somewhere condensed to spots fluorescence. And mutant 3a D1–77, D77–103, D99–147 and D99–274 were localized in the cytoplasm (G–O, S–U). However, construct 3a D147–274 gave rise to preservation of EGFP in the cytoplasm and nuclear (P–R). (D) Subcellular localization of deletant 3a protein with Golgi-DsRed protein. 293 cells co-transfected with the deletant 3a and Golgi/pDsRed-N1 plasmids. At 24 h after transfection, cells were fixed, and nuclear was stained with Hoechest. Deletant 3a-EGFP protein (green, 1, 5 and 9), Golgi-DsRed marker (red, 2, 6 and 10) and merge image (3, 7 and 11) were indicated.

Fig. 3

Expression and cellular localization of truncated 3a protein. (A) DNA constructs of deletant 3a protein. The truncated SARS-CoV 3a proteins were cloned into pEGFP-N1 vector as C-terminal fusion to EGFP. The different mutants were constructed based on bioinformative studies of SARS-CoV 3a. Kozak consensus translation initiation site was indicated. The animo acid positions for 3a were given at the low (a, b and c represented the three transmembrane regions). C or N from localization represented the cytoplasm or nuclear localization. (B) Western blotting analysis. 293 cells were transiently transfected with described plasmids separately. Cell lysates were prepared at 48 h post-transfection and separated by SDS-PAGE. Proteins transferred to PVDF membrane was probed with anti-GFP antibody. Sizes (kDa) of molecular mass markers were indicated on the left. (C) Cellular localization of truncated 3a protein. At 24 h post-transfection with indicated mutants, 293 cells were analyzed by confocal fluorescence microscopy. Left row (green) showed fusion EGFP fluorescence; middle row (red) showed PI stained cell nuclei; right row (yellow) represented overlapping green and red fluorescence. The fluorescent pattern of 3a D1–147, D1–103 and D77–274 (A–F, V–X), like wild-type 3a, was distributed in the cytoplasm and somewhere condensed to spots fluorescence. And mutant 3a D1–77, D77–103, D99–147 and D99–274 were localized in the cytoplasm (G–O, S–U). However, construct 3a D147–274 gave rise to preservation of EGFP in the cytoplasm and nuclear (P–R). (D) Subcellular localization of deletant 3a protein with Golgi-DsRed protein. 293 cells co-transfected with the deletant 3a and Golgi/pDsRed-N1 plasmids. At 24 h after transfection, cells were fixed, and nuclear was stained with Hoechest. Deletant 3a-EGFP protein (green, 1, 5 and 9), Golgi-DsRed marker (red, 2, 6 and 10) and merge image (3, 7 and 11) were indicated.

Fig. 3

Expression and cellular localization of truncated 3a protein. (A) DNA constructs of deletant 3a protein. The truncated SARS-CoV 3a proteins were cloned into pEGFP-N1 vector as C-terminal fusion to EGFP. The different mutants were constructed based on bioinformative studies of SARS-CoV 3a. Kozak consensus translation initiation site was indicated. The animo acid positions for 3a were given at the low (a, b and c represented the three transmembrane regions). C or N from localization represented the cytoplasm or nuclear localization. (B) Western blotting analysis. 293 cells were transiently transfected with described plasmids separately. Cell lysates were prepared at 48 h post-transfection and separated by SDS-PAGE. Proteins transferred to PVDF membrane was probed with anti-GFP antibody. Sizes (kDa) of molecular mass markers were indicated on the left. (C) Cellular localization of truncated 3a protein. At 24 h post-transfection with indicated mutants, 293 cells were analyzed by confocal fluorescence microscopy. Left row (green) showed fusion EGFP fluorescence; middle row (red) showed PI stained cell nuclei; right row (yellow) represented overlapping green and red fluorescence. The fluorescent pattern of 3a D1–147, D1–103 and D77–274 (A–F, V–X), like wild-type 3a, was distributed in the cytoplasm and somewhere condensed to spots fluorescence. And mutant 3a D1–77, D77–103, D99–147 and D99–274 were localized in the cytoplasm (G–O, S–U). However, construct 3a D147–274 gave rise to preservation of EGFP in the cytoplasm and nuclear (P–R). (D) Subcellular localization of deletant 3a protein with Golgi-DsRed protein. 293 cells co-transfected with the deletant 3a and Golgi/pDsRed-N1 plasmids. At 24 h after transfection, cells were fixed, and nuclear was stained with Hoechest. Deletant 3a-EGFP protein (green, 1, 5 and 9), Golgi-DsRed marker (red, 2, 6 and 10) and merge image (3, 7 and 11) were indicated.

Fig. 4

Membrane association of 3a protein. (A) Analysis of cytoplasmic and membrane fractions. Cells transfected with the pEGFP-N1 or 3a/pEGFP-N1 were harvested at 48 h post-transfection, and lyzed in Dounce homogenizer. Cytoplasmic (supernatant, C) and membrane (pellet, P) were prepared and analyzed by Western blotting with anti-GFP antibody. Sizes (kDa) of molecular mass markers were indicated on the right. (B) Mode of membrane association. At 48 h after transfection with 3a/pEGFP-N1, cells membrane fractions were separately extracted with the indicated buffer and centrifuged to obtain the pellet (in the top) and supernatant fractions (in the low), and then analyzed by Western blotting with anti-GFP antibody.

Fig. 5

Co-localization of 3a and M proteins. (A) Cellular localization of M protein. 293 cells were transiently transfected with M/pDsRed-N1 and M/pCMV-myc separately. Localization of the protein was observed by fluorescence confocal microscopy. Red fluorescence came from expressed M-DsRed protein, and green fluorescence was labeled by FITC conjugated goat anti-mouse antibody to show the expression of myc-M. The nuclear DNA was stained with Hoechest (blue). All three panels of a row had the same field of view. (B) Subcellular localization of M protein. The cells transfected with M/pCMV-myc and Golgi/pDsRed-N1 were labeled with FITC conjugated antibody to show the expression of myc-M, and nuclear DNA was stained with Hoechest. The merged image was represented region of overlap between the myc antibody (green-) and Golgi-DsRed (red-) image. All four panels of a row had the same field of view. (C) Co-localization analysis of 3a and M proteins. The cells transfected with 3a/pEGFP-N1 and M/pDsRed-N1 or 3a/pEGFP-N1 and M/pCMV-myc were analyzed by confocal fluorescence microscopy. The merged image was represented region of overlap between the 3a-EGFP (green-) and M-DsRed (red-) or 3a-EGFP (green-) and myc-M (red-) image. All four panels of a row had the same field of view.