Zn 2+ Binding to Cysteine-Rich Domain of Extracellular Human Immunodeficiency Virus Type 1 Tat Protein Is Associated with Tat Protein-Induced Apoptosis

The Tat protein has several functional domains, one of which is the cysteine-rich domain that is a highly conserved region in spite of the presence of many subtypes of human immunodeficiency virus type 1 (HIV-1). Although the cysteine-rich domain is a potential site for Zn²⁺ binding, it is controversial whether Zn²⁺ is substantially essential for the structure and activities of the Tat protein. To study the significance of Zn²⁺ in the cysteine-rich domain of the Tat protein particularly released to the extracellular space, we raised the monoclonal antibody (MAb) 5A4, which has an attractive property of recognizing the Zn²⁺-binding Tat_20-41 peptide but not the apo-Tat_20-41 peptide. MAb 5A4 inhibited the trans-activation of the HIV long terminal repeat (LTR) in HeLa-CD4-LTR/β-gal cells induced by treatment with the recombinant Tat protein, indicating that MAb 5A4 can recognize the full-length Tat protein and inhibit its trans-activity. The antibody also inhibited the apoptosis of Jurkat cells induced by treatment with the released native-Tat-protein-containing supernatant from the culture of HIV-1_JRFL-infected cells. These results suggest that Zn²⁺, whose structure is closely associated with not only the trans-activation of HIV-LTR but also the induction of apoptosis, binds to the extracellular native Tat protein. The Zn²⁺-binding cysteine-rich domain therefore can be a molecular target in the development of an anti-Tat vaccine and agents for the control of extracellular-Tat-protein-mediated pathogenesis leading to the progression of acquired immunodeficiency syndrome.