Effect of TAR mutations on gene expression and virus replication. (A) The LTR-tetO promoter region of HIV-rtTA with the original (TAR^m) or mutant (A to F) TAR sequence was placed upstream of the firefly luciferase gene. To determine dox responsiveness, C33A cells transfected with these plasmid reporter constructs and an rtTA-expression plasmid were cultured at different dox levels (0 to 1,000 ng/ml). A plasmid constitutively expressing Renilla luciferase was cotransfected to correct for differences in transfection efficiency, and the ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects the promoter activity. A construct with the wild-type HIV-1 LTR promoter (TAR^wt) was included as a control. Average values obtained in three transfections are shown, with the error bars indicating standard deviations. (B) SupT1 T cells were transfected with the original HIV-rtTA (TAR^m) and TAR-mutated variants (A to F) and cultured with 1 μg/ml dox for several weeks. CA-p24 levels in the culture supernatant were measured by ELISA. No virus replication was observed in the absence of dox (not shown). This experiment was repeated three times with similar results.

TAR mutations introduced in the dox-dependent HIV-rtTA. (A) A schematic of the HIV-rtTA genome is shown, with the LTR region subdivided into the U3, R, and U5 domains. Transcription starts at the first nucleotide of the 5′ R region, and the RNA transcripts are polyadenylated at the last nucleotide of the 3′ R region. Both the 5′ and the 3′ ends of the RNA molecule can fold a TAR and poly(A) hairpin. In the HIV-rtTA virus, the Tat-TAR axis of transcription regulation has been inactivated by mutation of both Tat and TAR (tat^m and TAR^m; crossed boxes). Transcription and replication of the virus was made dox dependent by the introduction of tetO elements in the U3 promoter region and by replacing the Nef gene with the rtTA gene. (B) HIV-rtTA is based on the HIV-1 molecular clone LAI, which contains a 57-nt wild-type TAR hairpin (TAR^wt). In HIV-rtTA, TAR was inactivated by nucleotide substitutions in both the bulge and loop motifs (TAR^m). The TAR^m sequence was partially or nearly completely deleted (mutants A to F). The deleted nucleotides are boxed in gray. All mutations were introduced in both the 5′ and 3′ LTR of HIV-rtTA.

Evolutionary repair of a hairpin structure at the 5′ end of the viral RNA. (A) The TAR-deleted E virus was cultured for up to 168 days (cultures I and II) or 97 days (culture III). Cellular proviral DNA was isolated at 104 and 168 days (culture I), at 99 and 168 days (culture II), or at 97 days (culture III), and the LTR region was subsequently PCR amplified and cloned into the TA-cloning vector. The nucleotide sequence of the TAR region was determined for three to seven clones for each sample. The −10-to-+78 region (with +1 indicating the transcription initiation site) is shown for the original HIV-rtTA (TAR^m), the E mutant, and the evolved viruses (with the frequency at which each sequence is observed [#] indicated on the left). Nucleotide substitutions, insertions, and deletions (Δ) are boxed in gray. Arrows indicate the duplicated R sequence observed in culture III. At the right, ER1, ER2, and ER3 indicate the evolved sequences recloned into the HIV-rtTA virus. (B) To assay replication of the original HIV-rtTA (TAR^m), the E mutant, and the evolved variants (ER1, ER2, and ER3), SupT1 T cells were transfected with the proviral constructs and cultured with 1 μg/ml dox. This experiment was repeated two times with similar results. (C) Secondary structure of the 5′ end of the viral RNA transcripts. (D) Determination of the transcription initiation site. C33A cells were transfected with HIV-rtTA proviral clones carrying the mutant E or the evolved ER1, ER2, or ER3 sequence. After 2 days of culturing with 1 μg/ml dox, intracellular RNA was isolated and decapped, and the 5′-terminal sequence of the viral RNA transcripts was analyzed by 5′ RACE. The cDNA fragments were cloned in the TA-cloning vector, and 5 to 11 clones were sequenced for each sample. The transcription start site (>) observed for each clone is shown on the corresponding proviral U3-R sequence (*; transcription of the E mutant may have started one nucleotide downstream of the indicated position, because the corresponding RNA sample had not been decapped prior to reverse transcription and RT may have copied the cap-G nucleotide into cDNA) (17).