The human immunodeficiency virus type 1 promoter contains a CATA box instead of a TATA box for optimal transcription and replication

Abstract

The human immunodeficiency virus type 1 (HIV-1) transcriptional promoter contains a single polymorphism in the TATA box. Most subtypes contain the sequence TATAAGC, but subtype E and some recombinant AG strains have the sequence TAAAAGC. Based on mutagenesis studies of cellular RNA polymerase II (pol II) promoters, it has been proposed that the subtype E TATA box is nonfunctional due to the T-to-A substitution at the critical position 3. By means of transcription and virus replication assays, we demonstrate that the true TATA box motif within the viral long terminal repeat (LTR) promoter starts two nucleotides further upstream. Because of this realignment, subtype E has the sequence CATAAAA and all other subtypes have the sequence CATATAA. The polymorphism therefore has shifted from position 3 to position 5 and is no longer incompatible with efficient transcription according to rules determined for cellular pol II promoters. In addition, through sensitive competition experiments, we demonstrate that the CATA box of subtypes B and E can be improved for replication by the mutations 1T and 5T, respectively. The fact that the fitness of both subtype LTRs can be increased by specific point mutations in the CATA box suggests that the transcriptional promoter of HIV-1 is fine-tuned towards a suboptimal level of replication. However, this replication rate may be optimal in the in vivo context of an infected individual.

FIG. 1.

HIV-1 LTR promoter and CATA boxes of different subtypes. (A) Proviral genome of HIV-1 (not to scale). (B) Part of the LTR promoter and the consensus sequence of the TATA box with flanking nucleotides. The only variation within the TATA box is the T/A polymorphism at position 5 in subtype E versus all other subtypes. The HIV-1 TATA box is known as the TATAAGC motif (positions 3 to 9), but we demonstrate in this study that the motif is in fact CATATAA (positions 1 to 7).

FIG. 2.

Transcriptional activities of subtype B TATA box mutants. Shown are the relative luciferase activities of four TATA box mutant LTRs and the wt subtype B LTR. The activity of the wt TATA box sequence was arbitrarily set at 1 in assays without (A) and with (B) the transactivator protein Tat. Transfections were performed eight times in C33A and HeLa cells. Error bars indicate standard deviations.

FIG. 3.

Transcriptional activities of subtype E TATA box mutants. Shown are the relative luciferase activities of four TATA box mutant LTRs and the wt subtype E LTR. The activity of the wt TATA box sequence was arbitrarily set at 1 in assays without (A) and with (B) the transactivator protein Tat. Transfections were performed eight times in C33A and HeLa cells. Error bars indicate standard deviations.

FIG. 4.

Replication of TATA box mutant viruses in SupT1 cells. (A) Replication of the wt subtype B virus (LAI) and four TATA box mutants. (B) Replication of the hybrid LAI virus with the subtype E LTR and the 5T mutant.

FIG. 5.

Transcription is impaired through mutation of position 2 in the CATA box. Shown are six CATA box mutants in the subtype B and E contexts. C33A cells were transfected with the indicated LTR-luciferase plasmids and Tat plasmid. Luciferase activity was measured at 2 days posttransfection. All values were related to the wt activity, which was arbitrarily set at 1 in the subtype B (A) and subtype E (B) contexts. The significance of changes in promoter activity with respect to the “parental” CATA box was determined by using a paired t test (***, P < 0.001). Error bars indicate standard errors.

FIG. 6.

Replication is impaired through mutation of position 2 in the CATA box. (A) Replication of the subtype B virus with a wt TATA box and three subtype B TATA box mutants. (B) Replication of the subtype E LTR virus with a wt TATA box and three subtype E TATA box mutants.

FIG. 7.

The mutant TBP_AS rescues transcription of position 2 CATA box mutants. Shown are three CATA box mutants in the subtype B (A) and E (B) contexts. Control transfections (−) and cotransfections with TBP_wt and TBP_AS were performed. No significant differences were observed between control transfections and cotransfection with TBP_wt. There were significant differences only between cotransfections with TBP_AS and TBP_wt, which were determined by a paired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant). Error bars indicate standard errors.