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

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 singlechain 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 Tat⅐TAR 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.

Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference Directed against Human Transcription Elongation Factor P-TEFb (CDK9/CyclinT1)

“…Most significantly, RNAi of P-TEFb resulted in a decrease in both Tat transactivation and HIV replication, suggesting that the amount of P-TEFb in cells is important for the fidelity of Tat transactivation and HIV replication. Previous studies, includ-ing P-TEFb immunodepletion analyses and studies with smallmolecule inhibitors of P-TEFb or anti-hCycT1 intrabodies, also supported an important role for P-TEFb in Tat transactivation and HIV replication (1,4,11,14,30). However, none of these studies specifically showed the effects of altering PTEFb protein levels in vivo.…”

Gene therapy progress and prospects: Novel gene therapy approaches for AIDS

“…In this study, several scFv were able to inhibit HIV-1 replication when expressed as intrabodies in non-Hodgkin's T-cell lymphoma-derived Supt1 cells. 28 The question obviously arises whether such targets are reasonably considered in the context of normal primary cells. Appropriate studies in normal hematopoietic cells in the context of a normal immune system in higher mammals would be warranted prior to any human studies.…”

Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association

“…Some of them were cloned into pSyn1 vector (17,18), expressed in Escherichia coli XL1-Blue (Stratagene), and purified from the periplasmic fractions. The others were cloned into pET22b(ϩ) vector (Novagen), expressed in E. coli BL21(DE3) (Novagen), and purified from insoluble fraction of the inclusion bodies.…”