Repression of the HIV-1 5' LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors.
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.
Reynolds
Lindsey
L
Gendaq Ltd., Sangamo Biosciences, Inc., 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, United Kingdom.
Ullman
Christopher
C
Moore
Michael
M
Isalan
Mark
M
West
Michelle J
MJ
Clapham
Paul
P
Klug
Aaron
A
Choo
Yen
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D004247:DNA; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*; D016335:Zinc Fingers
D014157:Transcription Factors; D004247:DNA