Targeting of HIV-1 Tat traffic and function by transduction-competent single chain antibodies

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.