Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution

Upon long-term culturing of SIV-rtTA, additional nucleotide substitutions were observed in TAR that affect the structure of this RNA element but that do not restore Tat binding. We demonstrate that the bulge and loop mutations that we had introduced in the TAR element of SIV-rtTA to inactivate the Tat-TAR mechanism, shifted the equilibrium between two alternative conformations of TAR. The additional TAR mutations observed in the evolved variants partially or completely restored this equilibrium, which suggests that the balance between the two TAR conformations is important for efficient viral replication. Moreover, SIV-rtTA acquired mutations in the U3 promoter region. We demonstrate that these TAR and U3 changes improve viral replication in T-cell lines and macaque peripheral blood mononuclear cells (PBMC) but do not affect dox-control.The dox-dependent SIV-rtTA variant was optimized by viral evolution, yielding variants that can be used to test the conditionally live virus vaccine approach and as a tool in SIV biology studies and vaccine research.More than 20 years after the identification of human immunodeficiency virus (HIV) as the causative agent of AIDS, an effective HIV/AIDS vaccine remains elusive. All vaccine candidates thus far tested in human efficacy trials have failed to prevent HIV infection or suppress the viral load. In the experimental model system of pathogenic simian immunodeficiency virus (SIV) in macaques, live attenuated virus vaccines have proven to be much more effective than other AIDS vaccine approaches. For example, 95% of the Indian rhesus macaques immunized with a live attenuated SIV demonstrated a viral load suppression of more than 3 logs (compared to unvaccinated animals) upon challenge with a wild-type SIV, whereas such protection was observed in only 7% of macaques immunized with other vaccine strategies [1]. In most of the studies, SIV was attenuated through deletion of one or several accessory functions from the viral genome (revi