NDLI: Inhibition of Tat-mediated Transactivation and HIV-1 Replication by Human Anti-hCyclinT1 Intrabodies

Content Provider	Journal of Biological Chemistry (JBC)
Author	Bai, Jirong Sui, Jianhua Zhu, Rui Ying Tallarico, Aimée St Clair Gennari, Francesca Zhang, Dongsheng Marasco, Wayne A.
Abstract	Human immunodeficiency virus, type 1 (HIV-1) replication requires the interaction of Tat protein with the human cyclinT1 (hCyclinT1) subunit of the positive transcription elongation factor (P-TEFb) complex, which then cooperatively binds to transactivation response element (TAR) RNA to transactivate HIV transcription. In this report, a non-immune human single-chain antibody (sFv) phage display library was used to isolate anti-hCyclinT1 sFvs that could disrupt hCyclinT1-Tat interactions. The N-terminal 272 residues of hCyclinT1, including the entire cyclin domains and the Tat·TAR recognition motif (TRM), that fully support Tat transactivation was used for panning, and of the five unique anti-hCyclinT1 sFvs that were obtained, three bound to the cyclin box domains and two bound to TRM. All sFvs could be expressed as intrabodies at high levels in transiently transfected 293T and in stable Jurkat and SupT1 transfectants and could specifically co-immunoprecipitate co-expressed hCyclinT1 in 293T cells with varying efficacy without disrupting hCyclinT1-Cdk9 interactions. In addition, two sFv clones (3R6-1 and 2R6-21) that mapped to the cyclin box domains markedly inhibited Tat-mediated transactivation in several transiently transfected cell lines without inhibiting basal transcription or inducing apoptosis. When HIV-1 challenge studies were performed on stable 3R6-1-expressing Jurkat T cells, near complete inhibition of viral replication was obtained at a low challenge dose, and 74–88% inhibition to HIV-1 replication was achieved at a high infection dose in SupT1 cells. These results provide proof-in-principle that anti-hCyclinT1 intrabodies can be designed to block HIV-1 replication without causing cellular toxicity, and as a result, they may be useful agents for “intracellular immunization”-based gene therapy strategies for HIV-1 infection/AIDS.
Related Links	http://www.jbc.org/content/278/3/1433.abstract
Ending Page	1442
Starting Page	1433
Page Count	10
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	3
Volume Number	278
DOI	10.1074/jbc.M208297200
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2003-01-17
Access Restriction	Open
Subject Keyword	Human immunodeficiency virus (HIV) Human cyclinT1 (hCyclinT1) Cyclin-dependent kinase 9 (Cdk9) Positive transcription elongation factor complex (P-TEFb) Transactivation response element (TAR) Tat·TAR recognition motif (TRM) Long terminal repeat (LTR) Single-chain antibody (sFv) Monoclonal antibody (mAb) Hemagglutinin (HA) Horseradish peroxidase (HRP) Glutathione S-transferase (GST) Phosphate-buffered saline (PBS) Enzyme-linked immunosorbent assay (ELISA) Cytomegalovirus (CMV) Nuclear localization signal (NLS) Nucleolar localization signal (NOL) Small nuclear RNA (snRNA) 50% tissue culture infectious dose (TCID50) GENES: STRUCTURE AND REGULATION
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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