Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions

Abstract

Integration of HIV-1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3' end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV-1 life cycle, substrate blockage of IN is an attractive strategy for therapeutic interference. In this study, we used the 2-LTR-circle junctions of HIV-1 DNA as a model to design zinc finger protein (ZFP) targeting at the end terminal portion of HIV-1 LTR. A six-contiguous ZFP, namely 2LTRZFP was designed using zinc finger tools. The designed motif was expressed and purified from E. coli to determine its binding properties. Surface plasmon resonance (SPR) was used to determine the binding affinity of 2LTRZFP to its target DNA. The level of dissociation constant (K(d)) was 12.0 nM. The competitive SPR confirmed that 2LTRZFP specifically interacted with its target DNA. The qualitative binding activity was subsequently determined by EMSA and demonstrated the aforementioned correlation. In addition, molecular modeling and binding energy analyses were carried out to provide structural insight into the binding of 2LTRZFP to the specific and nonspecific DNA target. It is suggested that hydrogen-bonding interactions play a key role in the DNA recognition mechanisms of the designed ZFP. Our study suggested an alternative HIV therapeutic strategy using ZFP interference of the HIV integration process.

Figure 1

Schematic representation of the circle junction region of HIV-1. Predicted organization of U3, R, and U5 in 2-LTR-circle junctions shown above, whereas sequence in box was selected for designing the ZFP.

Figure 2

(A) Full-length amino acid sequence of 2LTRZFP. The amino acids shown by underline were located at the specific positions of −1, 3, and 6 from the N-terminal of the α-helix, respectively. (B) Full-length optimized nucleotide sequence of 2LTRZFP. (C) Primary sequence alignments of Aart (PDB code 2I13) and 2LTRZFP with 80% sequence identity. The underlined letters represent amino acid residues of the ZFP those are different from the Aart peptide. Positions −1, 3, and 6 above the recognition helix column represent amino acid residues often involved in DNA recognition. Numbers at the end of each line show the running number of the last amino acid residue.

Figure 3

(A) SDS-PAGE analysis of His6-2LTRZFP-GFP at different and purification steps. Lane 1: protein marker, lane 2: total bacterial lysate, lane 3: total bacterial lysate with pTriEx-4-2LTRZFP-GFP after IPTG induction for overnight, lane 4: pass through lysate, lanes 5, 6: solution after washing the column with binding buffer and washing buffer, respectively, lane 7: elution of purified His6-2LTRZFP-GFP. The arrow indicates the size of 50 kDa of His6-2LTRZFP-GFP. Numbers on left show size in kilo Dalton of protein ladder. (B) Western blot analysis of His6-2LTRZFP-GFP at different condition of protein expression. Lane 1: total bacterial lysate, lanes 2, 3: total bacterial lysate with pTriEx-4-2LTRZFP-GFP after IPTG induction for 4 h, and overnight, respectively. The arrow indicates the size of 50 kDa of His6-2LTRZFP-GFP. Numbers on left show size in kilo Dalton of protein ladder.

Figure 4

Expression of green fluorescent protein in HeLa cells 24 h after transfection. (A, B) Transfected with recombinant plasmid pTriEx-4-GFP. (C, D) Transfected with recombinant plasmid pTriEx-4-2LTRZFP-GFP.

Figure 5

Kinetic analysis between 2LTRZFP-GFP and its target DNA sequence. (A) Binding kinetic of 2LTRZFP-GFP (300, 200, and 100 nM), GFP, and BSA to the immobilized chip with specific ds-DNA. (B) For the competitive SPR, 2LTRZFP-GFP was incubated with nonbiotinylated ds-DNA of its target ds-DNA, and nonspecific ds-DNA in zinc buffer for 15 min before injection. (C) 2LTRZFP-GFP was injected to determine the binding activity with its target DNA sequence in zinc buffer containing 1 μM, 1 mM, and 10 mM of EDTA.

Figure 6

Electrophoretic mobility shift assay (EMSA). (A) The protein used for each reaction is 1 μM, whereas [0], no DNA duplex; [+], specific DNA duplex; [−], nonspecific DNA duplex. The gel was stained with SYBR® Green EMSA stain (B) or with SYPRO® Ruby EMSA stain (C). In panels B and C: lanes 1 to 9, the specific DNA duplex used for each reaction was 250 nM, whereas 2LTRZFP-GFP was varied, the concentration from 0, 0.125, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 μM, respectively. Lane 10, 2LTRZFP-GFP was used 3.0 μM, but no DNA duplex.

Figure 7

Comparisons of conformation of the recognition binding region in Zif1 (in left panel) and Zif2 (in right panel). The nucleotides are shown in a licorice model and the protein backbone in a cartoon diagram.

Figure 8

Possible hydrogen bonding between the zinc finger residues and the target DNA triplet within distance < 3.5 A° between H-bond donor and H-bond acceptor associated with the distance.