Conference on Retroviruses and Opportunistic Infections edition:10 location:Boston, MA, USA date:10-14 February 2003
Background: During HIV-1 reverse transcription, the central polypurine tract (cPPT) and the central termination sequence (CTS) are responsible for the creation of a plus strand overlap: the central DNA flap. Recently it was demonstrated that the central DNA flap of HIV-1 acts as a cis-determinant of HIV-1 nuclear import. Insertion of a fragment encompassing the cPPT and CTS into HIV-1 derived vectors resulted in a major improvement of lentiviral vector performance. The improved performance of these cPPT vectors has been attributed to facilitated nuclear import. No direct impact on transgene expression was reported. However, the inserted fragment originates from a region of the HIV-1 genome that displays enhancer activity. We have now analyzed the effect of the HIV-1 cPPT-CTS fragment on transgene expression using quantitative real-time PCR to precisely correlate the expression level to the amount of integrated vector.
Methods: Different cell lines were transduced with HIV-1 derived vectors containing the cPPT-CTS fragment in sense or antisense orientation. Parental vectors lacking the cPPT-CTS fragment were used in parallel. All vectors encoded for EGFP. Cells were passaged after transduction to dilute unintegrated vector DNA, aliquots were analysed by FACS for EGFP expression, and DNA was extracted for PCR analysis. In addition, transfection experiments with the different plasmids were performed and the impact of the cPPT-CTS in different lentiviral vector backbones was assessed.
Results: Using quantitative real-time PCR, we precisely correlated the expression level of EGFP to the amount of integrated vector. The presence of the cPPT-CTS fragment in sense or antisense orientation significantly increased the transgene expression per integrated vector copy. Since the DNA flap is only formed in the sense orientation, the DNA flap itself is not involved. The stimulation was only observed in the context of an integrated vector, since in transfection experiments no impact on transgene expression was demonstrated. The extent of the stimulation of transgene expression by the cPPT-CTS fragment was dependent on the specific vector backbone.
Conclusions: The cPPT-CTS fragment originated from a region of the HIV-1 genome that displays enhancer activity. Our data demonstrate that this fragment retains enhancer activity for transgene expression from HIV-1 derived vectors. Stimulation of gene expression represents a hitherto unknown functionality of the HIV-1 cPPT-CTS fragment with importance for HIV vectorology. Moreover, the described enhancer may function during HIV replication as a viral determinant ensuring a productive outcome after integration.