Vaccine 24 (2006) 3127–3136 Targeting of HIV-1 Tat traffic and function by transduction-competent single chain antibodies Dietmar M. Theisen a,∗ , Carola Pongratz a , Katja Wiegmann a , Francisco Rivero b , Oleg Krut a , Martin Kr ¨ onke a,c a Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19–21, 50935 Cologne, Germany b Institute for Biochemistry, University of Cologne, Goldenfelsstrasse 19–21, 50935 Cologne, Germany c Center for Molecular Medicine Cologne, University of Cologne, Goldenfelsstrasse 19–21, 50935 Cologne, Germany Received 8 October 2005; received in revised form 11 January 2006; accepted 19 January 2006 Available online 9 February 2006 Abstract Human immunodeficiency virus type 1-encoded Tat protein is a transactivating factor essentially required for viral replication. Tat binds specifically to the transactivation response RNA stem loop, which is formed at the 5 ′ end of all viral transcripts. The TAR binding motif of Tat also contains a protein transduction domain, PTD that mediates not only nuclear localization of Tat but is also capable of transducing cargo across cellular membranes. In order to target a Tat antagonist directly to the TAR binding site in the nucleus, we engineered a chimeric protein consisting of the Tat-derived PTD fused to the anti-Tat single chain antibody scFvtat1 that binds intracellularly to Tat. Recombinant scFvtat1–PTD TAT fusion antibody retained both, anti-Tat specificity and PTD TAT -mediated transduction-competence leading to its nuclear accumulation within living cells. Incubation of Jurkat T cells with scFvtat1–PTD TAT suppressed Tat-dependent transcription of a HIV-1 reporter gene by >80%. Transfection of a scFvtat1–PTD TAT expression plasmid in HEK293 cells resulted in diffuse cytoplasmic and nuclear expression. ScFvtat1–PTD TAT did not inhibit HIV-1 Tat translocation to the nucleus, yet showed increased inhibition of 78%, indicating a nuclear site of scFvtat1–PTD TAT action. Strikingly, the PTD TAT alone showed 55% inhibition in the HIV-1 luciferase reporter assay, indicating competition with HIV-1 Tat binding to the TAR element. The results of this study suggest that Tat traffic can only marginally be affected by anti-Tat antibodies and that effective inhibition of Tat function requires both competition with HIV Tat for TAR binding mediated by PTD TAT and steric hindrance mediated by the scFvtat1 moiety. © 2006 Elsevier Ltd. All rights reserved. Keywords: Tat protein; Transduction-competent; Single chain antibody 1. Introduction The 14kDa regulatory HIV-1 Tat protein is essential for virus replication and pathogenesis and thus represents an attractive target for therapeutic intervention. In infected cells, Tat recruits cellular transcription factors to the long terminal repeat (LTR), ultimately promoting transcriptional elonga- tion and viral gene expression. Tat binds specifically to a Abbreviations: HCV, hepatitis C virus; HRP, horseradish peroxidase; PTD TAT , protein transduction domain of the Tat protein of HIV-1; RT, reverse transcriptase; scFv, single chain antibody ∗ Corresponding author. Tel.: +49 221 478 7288; fax: +49 221 478 7283. E-mail address: dietmar.theisen@uk.uni-koeln.de (D.M. Theisen). bulged region in the transactivation response (TAR) RNA stem loop, which is formed at the 5 ′ end of all viral transcripts. Tat binds cooperatively to TAR and interacts specifically with human cyclinT1, which eventually results in the assembly of Tat-TAR with the positive transcription elongation factor (P-TEFb) [1]. In the absence of Tat, viral gene expression is severely compromised [2]. Through an unknown mecha- nism, Tat activates genes encoding transcription factors that may, in turn regulate chemokine [3,4] and chemokine recep- tor expression [5,6], resulting in the formation of a chemokine gradient that attracts monocytes and activated CD4 + T cells. Tat exerts also immuno-suppressive activities by inhibit- ing mitogen-, alloantigen-, and antigen-induced lymphocyte 0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.01.055