Strict control of human immunodeficiency virus type 1 replication by a genetic switch: Tet for Tat

Abstract

Live-attenuated human immunodeficiency virus type 1 (HIV-1) variants have shown great promise as AIDS vaccines, but continued replication can lead to the selection of faster-replicating variants that are pathogenic. We therefore designed HIV-1 genomes that replicate exclusively upon addition of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. These designer "HIV-rtTA" viruses replicate in a strictly dox-dependent manner both in a T-cell line and in primary blood cells, and the rate of replication can be fine-tuned by simple variation of the dox concentration. These HIV-rtTA viruses provide a tool to perform genetics, e.g., selection and optimization experiments, with the E. coli-derived Tet reagents in a eukaryotic background. Furthermore, such viruses may represent improved vaccine candidates because their replication can be turned on and off at will.

FIG. 1

Design of dox-dependent HIV-1 variants. (A) HIV-1 genome and modifications that were introduced to construct HIV-rtTA. Details of the nine-step construction are provided in Materials and Methods. In brief, we inactivated the TAR-Tat transcriptional axis (marked in red), which was replaced by the tetracycline-inducible tetO-rtTA system (marked in green). Inactivation of TAR and Tat is indicated by crosses through the motifs. The size of the HIV-rtTA genome is larger than that of HIV-1 (the maps are not drawn to scale). The RNA genome of HIV-1 LAI is 9,229 nt, and HIV-rtTA is either 9,767 nt (the SYS and SWS variants) or 9,875 nt (KYK and KWK variants). (B) TAR hairpin structure and inactivating mutations that were introduced in the bulge (triple-nucleotide substitution) and in the loop (two point mutations). This RNA target binds the Tat-cyclin T1 complex during transcriptional activation of the LTR promoter (53). Panel C provides some details of the tetO insertion in the LTR promoter. The U3 region of the wild-type LTR (left) encodes two NF-κB sites (□) and three Sp1 sites (○). The modified LTR (right) contains either six or eight tetO operators (▴) upstream of the Sp1 sites. The six tetO variant (mutant S) has no NF-κB sites, whereas both NF-κB sites are present upstream of the eight tetO operators (mutant K). The arrow marks the transcription start site at the U3-R border, which also denotes the 5′ border of the TAR RNA hairpin motif.

FIG. 2

dox-controlled replication of the HIV-rtTA viruses. The SupT1 T-cell line was electroporated with 10 μg of the indicated molecular clones, and cells were cultured without or with an increasing concentration of dox (0 to 1,000 ng/ml). Some of the cultures were stopped at day 5 because of massive HIV-induced syncytium formation and cell death. Virus production was measured by CA-p24 ELISA on culture supernatant samples.

FIG. 3

dox-dependent replication of HIV-rtTA viruses in primary cells. PBMCs were electroporated with the KWK (diamonds) and KYK (triangles) constructs (20 μg), and the cultures were maintained without (open symbols) or with (1,000 ng/ml) (solid symbols) dox. Fresh uninfected cells were added immediately after transfection and at day 6 postinfection. Virus production was measured by CA-p24 ELISA on culture supernatant samples.

FIG. 4

Replication of HIV-rtTA can be turned on and turned off. These experiments were performed with the SWS virus, but similar experiments have been performed with the other HIV-rtTA variants. We used the SWS virus (2,200 ng of CA-p24) to infect 6 × 10⁶ SupT1 cells at day 0. (A) Replication potential with 0, 100, and 1,000 ng of dox per ml. (B) Effect of delayed addition of dox (1,000 ng/ml) at day 3 after infection. (C) Infected cells were grown in 1,000 ng of dox per ml for 3 days, at which point the cells were washed and incubated in the absence or presence of dox. (D) Infected cells were maintained in the presence of dox and either the Pro inhibitor SQV (200 nM) or the RT inhibitor AZT (1 μM).