scholarly journals Zinc Finger Protein Designed to Target 2-Long Terminal Repeat Junctions Interferes with Human Immunodeficiency Virus Integration

2012 ◽  
Vol 23 (9) ◽  
pp. 932-942 ◽  
Author(s):  
Supachai Sakkhachornphop ◽  
Carlos F. Barbas ◽  
Rassamee Keawvichit ◽  
Kanlaya Wongworapat ◽  
Chatchai Tayapiwatana
Keyword(s):  
Human Immunodeficiency Virus ◽  
Long Terminal Repeat ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Terminal Repeat ◽  
Finger Protein ◽  
Immunodeficiency Virus ◽  
Virus Integration

scholarly journals OTK18, a zinc-finger protein, regulates human immunodeficiency virus type 1 long terminal repeat through two distinct regulatory regions

2007 ◽  
Vol 88 (1) ◽  
pp. 236-241 ◽  
Author(s):  
Masahide Horiba ◽  
Lindsey B. Martinez ◽  
James L. Buescher ◽  
Shinji Sato ◽  
Jenae Limoges ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Long Terminal Repeat ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Regulatory Elements ◽  
Terminal Repeat ◽  
Human Macrophages ◽  
Finger Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

It has previously been shown by our laboratory that OTK18, a human immunodeficiency virus (HIV)-inducible zinc-finger protein, reduces progeny-virion production in infected human macrophages. OTK18 antiviral activity is mediated through suppression of Tat-induced HIV-1 long terminal repeat (LTR) promoter activity. Through the use of LTR-scanning mutant vectors, the specific regions responsible for OTK18-mediated LTR suppression have been defined. Two different LTR regions were identified as potential OTK18-binding sites by an enhanced DNA–transcription factor ELISA system; the negative-regulatory element (NRE) at −255/−238 and the Ets-binding site (EBS) at −150/−139 in the LTR. In addition, deletion of the EBS in the LTR blocked OTK18-mediated LTR suppression. These data indicate that OTK18 suppresses LTR activity through two distinct regulatory elements. Spontaneous mutations in these regions might enable HIV-1 to escape from OTK18 antiretroviral activity in human macrophages.

scholarly journals Human Immunodeficiency Virus Type 1 Incorporated with Fusion Proteins Consisting of Integrase and the Designed Polydactyl Zinc Finger Protein E2C Can Bias Integration of Viral DNA into a Predetermined Chromosomal Region in Human Cells

2006 ◽  
Vol 80 (4) ◽  
pp. 1939-1948 ◽  
Author(s):  
Wenjie Tan ◽  
Zheng Dong ◽  
Thomas A. Wilkinson ◽  
Carlos F. Barbas ◽  
Samson A. Chow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Fusion Protein ◽  
Binding Site ◽  
Zinc Finger ◽  
Fusion Proteins ◽  
Zinc Finger Protein ◽  
Chromosomal Region ◽  
Finger Protein ◽  
Immunodeficiency Virus

ABSTRACT In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5′ untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.

scholarly journals Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites

2004 ◽  
Vol 78 (3) ◽  
pp. 1301-1313 ◽  
Author(s):  
Wenjie Tan ◽  
Kai Zhu ◽  
David J. Segal ◽  
Carlos F. Barbas ◽  
Samson A. Chow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Zinc Finger ◽  
Fusion Proteins ◽  
Zinc Finger Protein ◽  
Recognition Sequence ◽  
Finger Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In order to establish a productive infection, a retrovirus must integrate the cDNA of its RNA genome into the host cell chromosome. While this critical process makes retroviruses an attractive vector for gene delivery, the nonspecific nature of integration presents inherent hazards and variations in gene expression. One approach to alleviating the problem involves fusing retroviral integrase to a sequence-specific DNA-binding protein that targets a defined chromosomal site. We prepared proteins consisting of wild-type or truncated human immunodeficiency virus type 1 (HIV-1) integrase fused to the synthetic polydactyl zinc finger protein E2C. The purified fusion proteins bound specifically to the 18-bp E2C recognition sequence as analyzed by DNase I footprinting. The fusion proteins were catalytically active and biased integration of retroviral DNA near the E2C-binding site in vitro. The distribution was asymmetric, and the major integration hot spots were localized within a 20-bp region upstream of the C-rich strand of the E2C recognition sequence. Integration bias was not observed with target plasmids bearing a mutated E2C-binding site or when HIV-1 integrase and E2C were added to the reaction as separate proteins. The results demonstrate that the integrase-E2C fusion proteins offer an efficient approach and a versatile framework for directing the integration of retroviral DNA into a predetermined DNA site.

scholarly journals Identification and Analysis of Mot3, a Zinc Finger Protein That Binds to the Retrotransposon Ty Long Terminal Repeat (δ) in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (4) ◽  
pp. 1879-1890 ◽  
Author(s):  
Jon M. Madison ◽  
Aimée M. Dudley ◽  
Fred Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Long Terminal Repeat ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Independent Study ◽  
Terminal Repeat ◽  
Mrna Levels ◽  
Finger Protein ◽  
Partial Suppression

ABSTRACT Spt3 and Mot1 are two transcription factors of Saccharomyces cerevisiae that are thought to act in a related fashion to control the function of TATA-binding protein (TBP). Current models suggest that while Spt3 and Mot1 do not directly interact, they do function in a related fashion to stabilize the TBP-TATA interaction at particular promoters. Consistent with this model, certain combinations of spt3 and mot1 mutations are inviable. To identify additional proteins related to Spt3 and Mot1 functions, we screened for high-copy-number suppressors of the mot1 spt3inviability. This screen identified a previously unstudied gene,MOT3, that encodes a zinc finger protein. We show that Mot3 binds in vitro to three sites within the retrotransposon Ty long terminal repeat (δ) sequence. One of these sites is immediately 5′ of the δ TATA region. Although a mot3 null mutation causes no strong phenotypes, it does cause some mild phenotypes, including a very modest increase in Ty mRNA levels, partial suppression of transcriptional defects caused by a mot1 mutation, and partial suppression of an spt3 mutation. These results, in conjunction with those of an independent study of Mot3 (A. Grishin, M. Rothenberg, M. A. Downs, and K. J. Blumer, Genetics, in press), suggest that this protein plays a varied role in gene expression that may be largely redundant with other factors.

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