Berkhout Lab - Ben Berkhout - HIV AIDS research Laboratory

The system for tetracycline-inducible transcription developed by Bujard and coworkers has become a widely used tool to regulate the expression of a given gene in eukaryotic cells. In one of its most evolved versions, the system is based on the rtTA chimeric transcriptional activator, containing a modified DNA binding domain of the E. coli Tet repressor coupled to the transactivation domain of VP16. Binding of rtTA to its cis-element and subsequent transactivation are tightly regulated by the tetracycline analog doxycycline (dox).

Construction of the HIV-rtTA
We have introduced the gene encoding the rtTA transcriptional activator protein in the place of the 3’ Nef gene, and 8 copies of the tetO operator binding sites were introduced into the LTR promoter. Furthermore, we inactivated the natural viral transactivator Tat and its RNA cis-element, TAR.

Enlarged view

The HIV-rtTA replicates only in the presence of doxycycline.

Application in vaccine studies
This conditional HIV virus, the replication of which is critically dependent on dox, may be an ideal reagent for innovative vaccine approaches to AIDS. Conditional virus replication provides a unique safety feature for a live attenuated virus vaccine. The HIV-rtTA virus allows one to control replication to the extent that is needed for induction of the immune system, after which replication can be turned off by withdrawal of dox. If needed, virus replication can be turned on at a later moment as booster vaccination.

Relevant publications

Berkhout B, Verhoef K, Marzio G, Klaver B, Vink M, Zhou X, Das AT. Conditional virus replication as an approach to a safe live attenuated human immunodeficiency virus vaccine. J Neurovirol. 2002 Dec;8 Suppl 2:134-7.

Das AT, Zhou X, Vink M, Klaver B, Berkhout B. Conditional live virus as a novel approach towards a safe live attenuated HIV vaccine. Expert Rev Vaccines. 2002 Oct;1(3):293-301.

Berkhout B, Marzio G and Verhoef K; Control over HIV-1 replication by an antibiotic; a novel vaccination strategy with a drug-dependent virus (2002) Virus Research 82, 103-108.

Marzio G, Verhoef K, Vink M, and Berkhout B. In vitro evolution of a highly replicating, doxycycline-dependent HIV for application in vaccine studies. Proc Natl Acad Sci U S A. 2001 May 22;98(11):6342-7.

Berkhout B, Verhoef K, van Wamel JLB and Back N. 1999. Genetic instability of live, attenuated human immunodeficiency virus vaccine strains. Journal of Virology 73, 1138-1145.

Verhoef K, Marzio G, Hillen W, Bujard H and Berkhout B. 2001. Strict control of HIV-1 replication by a genetic switch: Tet for Tat. Journal of Virology, 75, 979-87.