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J. Biol. Chem., Vol. 281, Issue 25, 17084-17091, June 23, 2006

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The Genetic Stability of a Conditional Live HIV-1 Variant Can Be Improved by Mutations in the Tet-On Regulatory System That Restrain Evolution^*

From the Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because more quickly replicating pathogenic virus variants may evolve after vaccination. As an alternative vaccine approach, we have previously presented a doxycycline (dox)-dependent HIV-1 variant that was constructed by incorporating the tetracycline-inducible gene expression system (Tet-On system) into the viral genome. Replication of this HIV-rtTA variant is driven by the dox-inducible transcriptional activator rtTA and can be switched on and off at will. A large scale evolution study was performed to test the genetic stability of this conditional live vaccine candidate. In several long term cultures, we selected for HIV-rtTA variants that no longer required dox for replication. These evolved variants acquired a typical amino acid substitution either at position 19 or 37 in the rtTA protein. Both mutations caused rtTA activity and viral replication in the absence of dox. We designed a novel rtTA variant with a higher genetic barrier toward these undesired evolutionary routes. The corresponding HIV-rtTA variant did not lose dox control in long term cultures, demonstrating its improved genetic stability.

Received for publication, December 16, 2005 , and in revised form, April 7, 2006.

^* This work was supported by the Technology Foundation STW (the applied science division of the Netherlands Organisation for Scientific Research NWO and the technology program of the Ministry of Economic Affairs, Utrecht, the Netherlands). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 31-20-566-4822; Fax: 31-20-691-6531; E-mail: b.berkhout{at}amc.uva.nl.

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