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204 Effect of Rev Expression on SIVmac239 gp130 Env Genes in Mammalian Cells
D. D. Wu*, D. Chen, and R. Brown
Quality Biological Inc., Gaithersburg, MD, USA
Background: Transport of messages from the nucleus to the cytoplasm is an essential step in the retroviral life cycle. Simian immunodeficiency virus (SIV) and human immunodeficiency virus-1 (HIV) have developed a highly efficient mechanism, the Rev and Rev-responsive element (RRE) system, to regulate the expression of unspliced and incompletely spliced mRNAs. Previous studies have shown that the SIVmac239 env gene with a natural signal sequence had an extremely low level of expression and secretion in eukaryotic expression systems. In this study, we investigate the effect of Rev on the expression of SIVmac293 gp130 in Cos-7 cells and CHO cells.
Methods: The leader signal sequence (84 bp) of gp130 was deleted. The resulting 1,470-bp fragment was inserted and fused to appropriate vectors containing either tissue plasminogen activator leader signal sequence (t-PA) or the immunoglobulin secretion signal sequence. The vector without any leader sequence was used as the control. The identity and orientation of cloned fragments were confirmed by restriction mapping and DNA sequencing. The pREV, a plasmid encoding the Rev protein of HIV, and the individual plasmid were cotransfected into Cos-7 or CHO cells by the liposome method. Three days after the transfection, supernatant and cell lyses of each transfected cell population were examined for gp130 gene expression by Western-blot analysis, using a mouse anti-SIVgp120 monoclonal antibody (KK65).
Results: The results show that cotransfection of REV significantly enhanced expression of the SIVgp130 env gene in Cos-7 cells, but had only very limited effect in CHO cells. The increased SIVgp130 env gene expression was found to increase proportionally in both growth medium and lysed cells. No significant difference in the effect of REV was observed in cells transfected with the vectors constructed with different leader signals.
Conclusions: We examined the ability of Rev to drive the expression of SIVmac239 env from subgenomic constructs with different leader signals. Rev increased SIVgp130 env gene expression in Cos-7 cells, regardless of the leader signal of the gene. The increased amount of protein was detected in both secreted form and nonsecreted form. The effect of Rev was found to be unrelated to the leader signal of the gene. As reported previously in murine cell lines, the CHO cells appeared to be defective in Rev-RRE activity.
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