Repression of the HIV-1 5' LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors

Abstract

Zinc finger domains are small DNA-binding modules that can be engineered to bind desired target sequences. Functional transcription factors can be made from these DNA-binding modules, by fusion with an appropriate effector domain. In this study, eight three-zinc-finger proteins (ZFPs) that bound HIV-1 sequences in vitro were engineered into transcription repressors by linking them to the Krüppel-associated box (KRAB) repressor domain (KOX1). One protein, ZFP HIVB-KOX, which bound to a 9-bp region overlapping two Sp1 sites, was found to repress a Tat-activated 5' LTR cellular HIV-reporter assay to almost basal levels. A related six-finger protein, HIVBA'-KOX, was made to target all three Sp1 sites in the 5' LTR promoter and efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells. In contrast, a combination of two unlinked three-finger ZFPs, HIVA'-KOX and HIVB-KOX, which bind over the same region of DNA, resulted in less effective repression. Finally, HIVBA'-KOX was tested for its capacity to block viral replication in a cellular infection assay using the HIV-1 HXB2 strain. This ZFP was found to inhibit HIV-1 replication by 75% compared with control constructs, thus demonstrating the potential of this approach for antiviral therapy.

Figure 1

DNA target sites and the corresponding ZFPs engineered to bind the HIV-1 promoter. (a) DNA sequence from the HIV-1 (HXB2 strain) 5′ LTR (GenBank accession no. K03455). Nucleotides are numbered relative to the transcription start site (+1). The sites targeted by the ZFPs HIVA–HIVG are underlined. Binding sites for NF-κB and Sp1 are also highlighted (adapted from refs. and 38). (b) DNA recognition by the three-finger peptides in this study. Potential protein–DNA contacts are indicated by dotted arrows. The products shown are derived from two combinatorial libraries whose randomized amino acid positions are shown as circles (numbered relative to their helical positions). Each library binds a complementary DNA sequence, “HIJKLM” or “NOPQ,” so that when these subdomains are recombined they make a full-length protein that potentially binds all 10 bp of the DNA, “HIJKLMNOPQ,” as shown (12). (c) Selection of DNA-binding domains to recognize the HIV-1 promoter. (i) Nucleotide sequences from HIV-1 of the form 3′-HIJKLMNOPQ-5′ (see above), as recognized by clones HIVA to HIVG. (ii) Amino acid sequences of the helical regions from recombinant ZFPs that recognize HIV-1 sequences. The origin of the amino acids is indicated by shading Lib12 and Lib23 residues. HIVA and HIVA′ contain some WT Zif268 residues (underlined). (iii) Apparent K_d for the interaction of the ZFPs for their cognate sequences as measured by phage ELISA (7, 12).

Figure 2

CAT assays for HIV-1 5′ LTR activity in the presence of ZFP-KOX transcription factors. Controls were basal LTR activity (without Tat) and activated LTR activity (0.1 μg of Tat plasmid added). ZFP repressors were coexpressed with the LTR-CAT reporter plasmid (10 μg) and HIV-1 Tat. Results are shown for three-finger repressor proteins, combinations of two three-finger ZFPs, and six-finger proteins.

Figure 3

Luciferase assays for HIV-1 5′ LTR activity in human T cells in the presence or absence of Tat, PMA/PHA, and control vector pcDNA3.1(−), HIVBA′-KOX, or HIVBA′.

Figure 4

Luciferase assays for HIV-1 5′ LTR activity in human T cells in the presence or absence of Tat, PMA/PHA, and various ZFP repressor constructs (150 ng of plasmid). The control contained pcDNA3.1(−) in place of the ZFP expression plasmid.

Figure 5

Assays to demonstrate inhibition of HIV-1 replication. The bars show HIV-1 focus-forming units (FFU)/ml, and the line shows the level of HIV-1 reverse transcriptase (RT) in the culture supernatant. The negative control contains pcDNA3.1 alone (A), whereas positive controls contain pcDNA3.1 + CXCR4 (B) or TFZ-KOX + CXCR4 (C). HIVBA′-KOX was tested and showed a reduction in the number of foci and the levels of viral RT (D).