Targeting of HIV-1 Tat traffic and function by transduction-competent single chain antibodies.
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.
Theisen
Dietmar M
DM
Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19-21, 50935 Cologne, Germany. dietmar.theisen@uk.uni-koeln.de
Pongratz
Carola
C
Wiegmann
Katja
K
Rivero
Francisco
F
Krut
Oleg
O
Krönke
Martin
M
D020816:Amino Acid Motifs; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D015696:Gene Products, tat / Q000037:antagonists & inhibitors* / Q000276:immunology*; D017930:Genes, Reporter; D015483:HIV Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D015483:HIV Antibodies; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D008156:Luciferases