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 0 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.

Zinc Finger Protein Designed to Target 2-Long Terminal Repeat Junctions Interferes with Human Immunodeficiency Virus Integration

“…The 2LTRZFP-GFP and Aart-GFP gene fragments were amplified from pTriEx-4-2LTRZFP-GFP (as previously described; Sakkhachornphop et al, 2009) and pTriEx-4-Aart-GFP, respectively. Aart is a six-zinc finger protein designed to recognize a unique 18-bp target site not found in the human genome, and this gene was used as a control (Dreier et al, 2001).…”

“…Cells were adjusted to 1 • 10 6 cells/ml in 5 ml of culture medium (RPMI 1640 medium supplemented with 10% FBS, interleukin-2 [10 U/ml], glutamine, and antibiotics) and stimulated with phytohemagglutinin (2 lg/ml; Sigma-Aldrich) in a humidified atmosphere with 5% CO 2 at 37°C. After 24 hr of incubation, 2 • 10 6 cells were mixed with 5 lg of plasmid vector (pTriEx-4-2LTRZFP-GFP [Sakkhachornphop et al, 2009], pTriEx-4-Aart-GFP, or pTriEx-4-GFP) and GTporator electroporation solution (Protean, Budějovice, Czech Republic), which was provided by Watchara Kasinrerk (Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand). The mix was electrotransfected, using program T-020 in the Nucleofector II device (Amaxa Biosystems).…”

“…This ZFP, called 2LTRZFP-GFP, binds to the integrase recognition sequence at 2-LTR circle junctions with nanomolar affinity, which is similar to the affinity of HIV-1 IN (Bugreev et al, 2003;Deprez et al, 2004). The GFP reporter confirmed that 2LTRZFP-GFP folds correctly in the cytoplasm and homes to the nucleus (Sakkhachornphop et al, 2009). In this study, we investigated whether 2LTRZFP-GFP interferes with IN activity during the formation of HIV-1 DNA noncovalent 2-LTR circle junctions.…”

“…We reported a designed Cys 2 His 2 ZFP fused with green fluorescent protein (GFP) that targets the terminal region of the HIV-1 LTR (Sakkhachornphop et al, 2009). This ZFP, called 2LTRZFP-GFP, binds to the integrase recognition sequence at 2-LTR circle junctions with nanomolar affinity, which is similar to the affinity of HIV-1 IN (Bugreev et al, 2003;Deprez et al, 2004).…”

Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells

“…Owing to its relative simplicity, ZFN-mediated genome surgery in primary human cells has become a reality: two phase I clinical trials (NCT00842634; NCT01044654) are underway to determine whether destruction of the CCR5 gene can confer protection from HIV infection when ZFNs are used to target and destroy CCR5 in autologous T cells from HIV patients. There are also studies using zinc-finger proteins to act as repressors of HIV (23,24) and to interfere with integration (25,26). However, none of the current strategies can directly target integrated HIV provirus genomes for cleavage using ZFNs.…”