>>Published online before print February 6, 2003
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.252770699
Biochemistry
Repression of the HIV-1 5' LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors
Lindsey Reynolds *, Christopher Ullman *, Michael Moore *, Mark Isalan *¶, Michelle J. West ||, Paul Clapham **, Aaron Klug , and Yen Choo *
*Gendaq Ltd., Sangamo Biosciences, Inc., 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, United Kingdom; Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom; ||School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom; and **Wohl Virion Centre, Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, 46 Cleveland Street, London W1T 4JF, United Kingdom
Contributed by Aaron Klug, December 18, 2002
Zinc finger domains are small DNA-binding modules that can be engineered to bind desired target sequences. Functional transcription factors can be made from these DNA-binding modules, by fusion with an appropriate effector domain. In this study, eight three-zinc-finger proteins (ZFPs) that bound HIV-1 sequences in vitro were engineered into transcription repressors by linking them to the Krüppel-associated box (KRAB) repressor domain (KOX1). One protein, ZFP HIVB-KOX, which bound to a 9-bp region overlapping two Sp1 sites, was found to repress a Tat-activated 5' LTR cellular HIV-reporter assay to almost basal levels. A related six-finger protein, HIVBA'-KOX, was made to target all three Sp1 sites in the 5' LTR promoter and efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells. In contrast, a combination of two unlinked three-finger ZFPs, HIVA'-KOX and HIVB-KOX, which bind over the same region of DNA, resulted in less effective repression. Finally, HIVBA'-KOX was tested for its capacity to block viral replication in a cellular infection assay using the HIV-1 HXB2 strain. This ZFP was found to inhibit HIV-1 replication by 75% compared with control constructs, thus demonstrating the potential of this approach for antiviral therapy.<<
>>Published online before print February 6, 2003
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.252773399
Biochemistry
Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors
Monika Papworth *, Michael Moore *, Mark Isalan *, Michal Minczuk *, Yen Choo *, and Aaron Klug *
*Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom; and Department of Genetics, Warsaw University, Pawinskiego 5A, 02-106 Warsaw, Poland
Contributed by Aaron Klug, December 18, 2002
The herpes simplex virus 1 (HSV-1) replicative cycle begins by binding of the viral activator, VP16, to a set of sequences in the immediate-early (IE) gene promoters. With the aim of inhibiting this cycle, we have constructed a number of synthetic zinc-finger DNA-binding peptides by using recently reported methods. Peptides containing either three or six fingers, targeted to a viral promoter, were engineered as fusions with a KOX-1 transcription repression domain. These proteins bound to the HSV-1 IE175k (ICP4) promoter, in vitro, with nanomolar or subnanomolar binding affinity. However, in a chloramphenicol acetyltransferase reporter system, only the six-finger protein was found to repress VP16-activated transcription significantly. Thus the longer array of zinc fingers is required to compete successfully against VP16, one of the most powerful natural activators known. We found that the HSV-1 replication cycle can be partially repressed by the six-finger peptide with the viral titer reduced by 90%.<<
Cheers, Tuck |
