Evolution of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Promoter by Conversion of an NF-κB Enhancer Element into a GABP Binding Site

Abstract

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein and cellular factors, of which the concentration and activity may depend on the cell type. Viral long terminal repeat (LTR) promoter sequences are therefore optimized to suit the specific nuclear environment of the target host cell. In long-term cultures of a Tat-defective, poorly replicating HIV-1 mutant, we selected for a faster-replicating virus with a 1-nucleotide deletion in the upstream copy of two highly conserved NF-κB binding sites. The variant enhancer sequence demonstrated a severe loss of NF-κB binding in protein binding assays. Interestingly, we observed a new binding activity that is specific for the variant NF-κB sequence and is present in the nuclear extract of unstimulated cells that lack NF-κB. These results suggest that inactivation of the NF-κB site coincides with binding of another transcription factor. Fine mapping of the sequence requirements for binding of this factor revealed a core sequence similar to that of Ets binding sites, and supershift assays with antibodies demonstrated the involvement of the GABP transcription factor. Transient transfection experiments with LTR-chloramphenicol acetyltransferase constructs indicated that the variant LTR promoter is specifically inhibited by GABP in the absence of Tat, but this promoter was dramatically more responsive to Tat than the wild-type LTR. Introduction of this GABP site into the LAI virus yielded a specific gain of fitness in SupT1 cells, which contain little NF-κB protein. These results suggest that GABP potentiates Tat-mediated activation of LTR transcription and viral replication in some cell types. Conversion of an NF-κB into a GABP binding site is likely to have occurred also during the worldwide spread of HIV-1, as we noticed the same LTR modification in subtype E isolates from Thailand. This typical LTR promoter configuration may provide these viruses with unique biological properties.

Regulation of HIV-1 gene transcription: from lymphocytes to microglial cells

“…Some subtypes of HIV-1, such as C, E, and A, appear to be transmitted more efficiently than the B clade. Variations in the nucleotide sequence of the LTR isolated from different HIV-1 M subtypes and strains have been reported [13,[167][168][169][170]. This LTR variability may have resulted from adaptation to specific cellular backgrounds to optimize HIV-1 gene transcription.…”

“…Recently, a variant of a poorly replicative virus has been described in SupT1 cells [169]. The variant LTR sequence presents a severe loss of NF-B binding directly linked to an adaptative point mutation in the second NF-B-binding sequence.…”

Human Immunodeficiency Virus Type 1 Subtype C Exhibits Higher Transactivation Activity of Tat than Subtypes B and E

“…This basic domain was shown to confer the trans-activation responsive (TAR) RNA binding properties of Tat (26). Compared with the sequences of wild-type B-and E-Tat at the downstream of this basic domain, the C-Tat has further several variations such as Glu 63 . The Ser 57 and Glu 63 were highly conserved among subtype C isolates reported previously (Fig.…”

“…Therefore, we next tested the subtype C-Tat specific amino acid substitution in comparison to that in subtypes B and E Tat at regions close to Ser 57 . Interestingly, Gln63 to Glu 63 is identified as a highly conserved amino acid residue among subtype C Tat proteins reported before(15,32,38,49) and was further characterized as a candidate responsible for the C-Tat activity. In fact, both of the mutant Tat proteins with substitution from Arg 57 to Ser57 and Gln 63 to Glu 63 in subtype B and Ser 57 to Arg57 and Glu 63 to Gln 63 in subtype C were significantly different from their original wildtype Tats in their activities.…”

“…This difference was shown by transfection assay in HeLa cells to lead to higher promoter/enhancer activities of subtype C LTR (C-LTR) than those of the other subtypes such as B and E (45). However, transcription factors other than NF-κB were also shown to be differently involved in the transactivation from the LTRs among the three subtypes, as shown in E-LTR in which GABP protein and Ets family transcription factor are active at the mutation site of NF-κB (63). In addition, there is also the subtype-specific loss or gain of motifs, such as the upstream stimulating factor (USF) specific to subtype B, and the number of AP-1 sites varying from zero to two for the different subtypes (25,44).…”