title: Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration.
abstract: Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine-adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future.
authors.affiliation: Division of Clinical Immunology, Department of Medical Technology, Chiang Mai University, Chiang Mai, Thailand.
authors.forename: Supachai
authors.initials: S
authors.lastname: Sakkhachornphop
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
authors.forename: Rassamee
authors.initials: R
authors.lastname: Keawvichit
authors.forename: Kanlaya
authors.initials: K
authors.lastname: Wongworapat
authors.forename: Chatchai
authors.initials: C
authors.lastname: Tayapiwatana
mesh_terms: D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013601:T-Lymphocytes / Q000378:metabolism* / Q000821:virology; D014161:Transduction, Genetic; D016662:Virus Integration; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins