AEGiS-15IAC: Targeting HIV-1 Tat protein to the virion.

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Targeting HIV-1 Tat protein to the virion.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. TuOrA1136)

Frankel FA, Roldan HA, Liang C, Wainberg MA
Jewish General Hospital - LDI. McGill University, Montreal, Canada

BACKGROUND: HIV-1 Tat is a small basic protein which is synthesized from multiply spliced viral RNA and is required for both efficient viral gene expression and reverse transcription. The presence of Tat in the virion has never been reported, yet Tat is able to exert effects on reverse transcription in a new round of infection. Thus, Tat may perform this function before virus budding or may be modified in the virion by several factors. We wished to assess whether the addition of a tag to the N-terminal domain of Tat would affect viral replication and to evaluate whether Tat protein is incorporated into the viral particle.

METHODS: To address those issues, we constructed a series of Vpr-Tat fusion proviruses and tagged-Tat constructs to be used in transfection and infection assays. The presence of HIV-1 Tat was studied in transfected COS-7 cells and in highly purified viral particles by immunochemical techniques.

RESULTS: Vpr-Tat fusion proviruses did not show differences with wild type provirus in regard to viral replication in cell culture assays. Hemmaglutinin-tagged Tat provirus did not seem to have affected viral replication. However, Flag-tagged Tat provirus did not grow at all. Both Hemmaglutinin and Flag Tagged-Tat were detectable in cell lysates. However, they were not detectable in highly purified viruses.

CONCLUSIONS: This study shows that Flag-tagged Tat is not biologically active. This is probably due to interactions between the Aspartic acid-rich N-terminal domain of Flag with the basic domain of Tat. Our data did not show the presence of Tat in the virion. However, the fact that Tagged-Tat protein would be cleaved by HIV protease would lead to either their exclusion from the virion or incorporation of modified forms which are not detectable by current available antibodies.

Keywords: AEGIS, Gene Products, tat, Reverse Transcription, Virion, HIV-1, Genes, tat, HIV Protease, Virus Replication, Proviruses, RNA, Viral, Genes, vpr, Transfection, Genes, Viral, genetics, virology

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TuOrA1136