Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome-associated coronavirus

Abstract

SARS 8b is one of the putative accessory proteins of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) with unknown functions. In this study, the cellular localization and activity of this estimated 9.6 kDa protein were examined. Confocal microscopy results indicated that SARS 8b is localized in both nucleus and cytoplasm of mammalian cells. Functional study revealed that overexpression of SARS 8b induced DNA synthesis. Coexpression of SARS 8b and SARS 6, a previously characterized SARS-CoV accessory protein, did not elicit synergistic effects on DNA synthesis.

Figure 1

Expression of recombinant SARS 8b‐EGFP protein in Vero E6 and CHO cells. Total cell proteins were extracted and 50–150 μg of protein were resolved by 12% SDS–PAGE. Western blots of these proteins were probed with anti‐GFP antibodies. Lane 1: 50 μg of proteins from Vero E6 cells transfected with pEGFP vectors. Lane 2: 150 μg of proteins from Vero E6 cells transfected with SARS 8b‐EGFP vectors. Lane 3: 50 μg of proteins from Vero E6 cells transfected with SARS 8b‐EGFP vectors. Lane 4: 30 μg of proteins from CHO cells transfected with pEGFP vectors. Lane 5: 30 μg of proteins from CHO cells transfected with SARS 8b‐EGFP vectors.

Figure 2

Subcellular localization of individually expressed recombinant SARS 8b‐EGFP and SARS 6‐RFP proteins. Images shown are Vero E6 cells transfected with vectors encoding (A) EGFP, (B) SARS 8b‐EGFP, and (C) SARS 6‐RFP; and CHO cells transfected with vectors encoding (D) EGFP, (E) SARS 8b‐EGFP, and (F) SARS 6‐RFP.

Figure 3

Subcellular localization of co‐expressed SARS 8b‐EGFP and SARS 6‐RFP proteins. Localization of cotransfected SARS 8b‐EGFP and SARS 6‐RFP in Vero E6 cells: (A) SARS 8b‐EGFP, (B) SARS 6‐RFP, (C) overlay image of (A) and (B). Localization of cotransfected SARS 8b‐EGFP and SARS 6‐RFP in CHO cells: (D) SARS 8b‐EGFP, (E) SARS 6‐RFP, (F) overlay image of (D) and (E).

Figure 4

Expression of untagged SARS 6 and SARS 8b mRNAs in Vero E6 cells. RT‐PCR was performed with β‐actin, SARS 6, and/ or SARS 8b primers on mRNAs extracted from control cells and cells transiently transfected with SARS 6 and/or SARS 8b vectors. PCR products for SARS 6, SARS 8b, β‐actin were 192, 255, and 481 bp, respectively.

Figure 5

Stimulation of DNA synthesis by untagged SARS 8b expression. [³H]‐thymidine incorporation assays were performed on (A) Vero E6 cells and (B) CHO cells transiently transfected with vector control and SARS 8b vectors. Each bar represents the means ± S.D. of three experiments in four‐ to six‐replicate setup. ^∗Significant difference between the control and SARS 8b‐expressing cells detected by Student's t test (p < 0.05).

Figure 6

Stimulation of DNA synthesis by untagged SARS 6 and SARS 8b expression. [³H]‐thymidine incorporation assays were performed on (A) Vero E6 cells and (B) CHO cells transiently transfected with vector controls, SARS 6 and/or SARS 8b, respectively. Each bar represents the means ± S.D. of three to five experiments in four‐ to six‐replicate setup. ^∗Significant differences among the controls and SARS proteins‐expressing cells detected by Kruskal–Wallis ANOVA on Ranks (p < 0.05).