8th International AIDS Conference Amsterdam, Netherlands — July 19-24, 1992

Int Conf AIDS 1992 Jul 19-24; 8:We46 (abstract no. WeA 1009)
Koken S, van Wamel J, Goudsmit J, Geelen JL, Berkhout B; Human Retrovirus Laboratory, UvA, Amsterdam.

OBJECTIVES: The HIV-1 LTR functions as the transcriptional promoter and contains multiple regulatory elements. Variation within these elements may have profound effects on the viral replication rate and viral pathogenicity. The goal of this study was to detect and analyze naturally occurring length variations in the HIV-1 promoter region.

METHODS: DNA was isolated from peripheral blood mononuclear cells (PBMC) of 16 HIV-1 infected individuals at different stages of disease, and from brain and spleen tissues of an HIV infected child. A PCR of the HIV-1 LTR promoter region was used to identify viruses with aberrant LTR structures. Promoter activity of the LTR variants was assayed upon transfection of LTR-CAT constructs into different cell lines. In order to measure viral replication rates, the different LTRs were introduced into an HIV-1 molecular clone and transfected into different cell lines. Virus production was monitored by measuring gag-p24 antigen and RT activity in the culture supernatant.

RESULTS: Two classes of LTR size variants were found. When compared to the wildtype HXB2 isolate either an additional Sp1 binding site or a duplication of a short DNA motif upstream of the NF-kB site was detected. The natural variant with 4 Sp1 sites showed a slightly higher promoter activity and viral replication rate than the wildtype LTR with 3 Sp1 sites. In contrast, there was no measurable positive effect of the duplicated motif. Nevertheless, this region does contribute to promoter activity, since deletion of the sequence results in reduced promoter activity. In order to measure accurately differences in virus production, an equal amount of an LTR size variant and the isogenic control plasmid was co-transfected into T-cell lines. A small defect in LTR promoter function of one virus is expected to result in outgrowth of the other. This co-transfection procedure resulted in the outgrowth of viruses with 4 Sp1 sites and viruses with 1 copy of the duplicated motif in 35 and 42 days, respectively. A difference in virus production of approximately 5 to 10% was calculated from the rate of outgrowth of the viruses. The duplications found in the LTRs of 4 different patients all contained the short sequence motif 5'-ACTGCTGA-3'. Electrophoretic mobility shift assays and UV cross-linking experiments were used to analyze protein binding to this motif. These experiments showed that a family of proteins (50-60 kD) binds to this motif.

CONCLUSIONS: Variation in the LTR is detected in patients at different stages of disease. The LTR length polymorphism results from duplication of promoter-enhancer elements, being either an additional Sp1 site or a duplication of the 5'-ACTGCTGA-3' motif.

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