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 vector modificationslentiviral vector backbones was assessed.

Results: Using quantitative real-time PCR, we precisely correlated the expression level of EGFP to the amount of integrated vector. We observed that tThe presence of the cPPT-CTS fragment in sense of or antisense orientationsignificantly 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 impact extent of the stimulation of transgene expression by the cPPT-CTS fragment on transgene expression 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 indeed retains enhances enhancer activity for transgene gene 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. and may function as aMoreover, the described enhancer may function during HIV replication viral as a viral determinant ensuring a productive outcome after integration.Stimulation of gene expression represents a hitherto unknown functionality of the HIV-1 cPPT-CTS fragment.

The HIV-1 cPPT mediates efficient lentiviral expression and increases nuclear import in ectopic positions

 

Impact of the HIV-1 cPPT on gene expression and nuclear import

 

Johan van Griensven, Bénédicte Van Maele, Jan De Rijck, Erik De Clercq and Zeger Debyser

 

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. The exact role/function of the DNA flap is not determined yet. 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.((and several data point to the existence of viral mechanisms ensuring a productive outcome after integration.)) We have now analysed 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. To further characterise the DNA flap, we analysed the nuclear import and integration of vectors containing the DNA flap in ectopic positions by quantitative PCR.

 

Methods:

Different cell lines were transduced with HIV-1 derived vectors containing the cPPT-CTS fragment in sense or antisense orientation. We constructed vectors containing the cPPT-CTS fragment at the 3’ or 5’ end of the vector DNA. All vectors encoded EGFP. Aliquots were analysed by flow cytometry for EGFP expression and DNA was extracted for PCR analysis for circular vector DNA and integrated vector.

 

Results

Using quantitative real-time PCR, we precisely correlated the EGFP expression level to the amount of integrated vector. We observed that the presence of the cPPT-CTS fragment in sense of in 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 integrated vector DNA, since in transfection experiments no impact on transgene expression was demonstrated. In addition, we observed that the increased nuclear import mediated by the DNA flap is independent of position in the vector DNA.

Conclusions

The cPPT-CTS fragment originated from a region of the HIV-1 genome that displays enhancer activity. Our data demonstrate that this fragment indeed enhances gene expression from HIV-1 derived vectors and may function as a viral determinant ensuring a productive outcome after integration. Stimulation of gene expression represents a hitherto unknown functionality of the HIV-1 cPPT-CTS fragment. The function of the DNA flap is independent of the position, pointing to a role as…