Attenuation of HIV-1 Replication in Primary Human Cells with a Designed Zinc Finger Transcription Factor*

Small molecule inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been extremely successful but are associated with a myriad of undesirable effects and require lifelong daily dosing. In this study we explore an alternative approach, that of inducing intracellular immunity using designed, zinc finger-based transcription factors. Three transcriptional repression proteins were engineered to bind sites in the HIV-1 promoter that were expected to be both accessible in chromatin structure and highly conserved in sequence structure among the various HIV-1 subgroups. Transient transfection assays identified one factor, KRAB-HLTR3, as being able to achieve 100-fold repression of an HIV-1 promoter. Specificity of repression was demonstrated by the lack of repression of other promoters. This factor was further shown to repress the replication of several HIV-1 viral strains 10- to 100-fold in T-cell lines and primary human peripheral blood mononuclear cells. Repression was observed for at least 18 days with no significant cytotoxicity. Stable T-cell lines expressing the factor also do not show obvious signs of cytotoxicity. These characteristics present KRAB-HLTR3 as an attractive candidate for development in an intracellular immunization strategy for anti-HIV-1 therapy.

[1]  B. Cullen,et al.  Inhibition of Human Immunodeficiency Virus Type 1 Replication in Primary Macrophages by Using Tat- or CCR5-Specific Small Interfering RNAs Expressed from a Lentivirus Vector , 2003, Journal of Virology.

[2]  J. Sodroski,et al.  Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity , 1986, Nature.

[3]  Robert F. Siliciano,et al.  Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1 , 2000, Journal of Virology.

[4]  U. Schopfer,et al.  Chemically Regulated Zinc Finger Transcription Factors* , 2000, The Journal of Biological Chemistry.

[5]  Y. Cheng,et al.  Antisense oligonucleotides as therapeutic agents--is the bullet really magical? , 1993, Science.

[6]  E. Branch,et al.  Cellular and anatomical reservoirs of HIV-1 in patients receiving potent antiretroviral combination therapy. , 1998, JAMA.

[7]  K. Jeang,et al.  Increased spacing between Sp1 and TATAA renders human immunodeficiency virus type 1 replication defective: implication for Tat function , 1993, Journal of virology.

[8]  K. Jeang,et al.  Multifaceted Activities of the HIV-1 Transactivator of Transcription, Tat* , 1999, The Journal of Biological Chemistry.

[9]  B. Berkhout,et al.  Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat , 1992, Journal of virology.

[10]  David J Segal,et al.  The use of zinc finger peptides to study the role of specific factor binding sites in the chromatin environment. , 2002, Methods.

[11]  D J Segal,et al.  Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. Van Lint,et al.  Chromatin disruption in the promoter of human immunodeficiency virus type 1 during transcriptional activation. , 1993, The EMBO journal.

[13]  C. Barbas,et al.  Functional Neutralization of HIV-1 Vif Protein by Intracellular Immunization Inhibits Reverse Transcription and Viral Replication* , 2002, The Journal of Biological Chemistry.

[14]  D J Segal,et al.  Insights into the molecular recognition of the 5'-GNN-3' family of DNA sequences by zinc finger domains. , 2000, Journal of molecular biology.

[15]  D. Margolis,et al.  The regulation of HIV-1 gene expression: the emerging role of chromatin. , 2002, DNA and cell biology.

[16]  H. Friedman,et al.  An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1 , 1992, Journal of virology.

[17]  D J Segal,et al.  Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[18]  B. Cullen,et al.  Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference , 2002, Journal of Virology.

[19]  A. Wolffe,et al.  PPARγ knockdown by engineered transcription factors: exogenous PPARγ2 but not PPARγ1 reactivates adipogenesis , 2002 .

[20]  C C Case,et al.  Synthetic Zinc Finger Transcription Factor Action at an Endogenous Chromosomal Site , 2000, The Journal of Biological Chemistry.

[21]  H. Blau,et al.  Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  H. Gendelman,et al.  Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone , 1986, Journal of virology.

[23]  M. Martin,et al.  Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types , 1991, Journal of virology.

[24]  D. Baltimore.  Intracellular immunization , 1988, Nature.

[25]  Luigi Naldini,et al.  Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo , 1997, Nature Biotechnology.

[26]  D. Trono,et al.  Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery , 1998, Journal of Virology.

[27]  C. Barbas,et al.  Controlling gene expression in plants using synthetic zinc finger transcription factors. , 2002, The Plant journal : for cell and molecular biology.

[28]  C. Barbas,et al.  Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Pilar Blancafort,et al.  Scanning the human genome with combinatorial transcription factor libraries , 2003, Nature Biotechnology.

[30]  J R Desjarlais,et al.  Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Carl O. Pabo,et al.  Cellular uptake of the tat protein from human immunodeficiency virus , 1988, Cell.

[32]  R. Tjian,et al.  Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. , 1986, Science.

[33]  H. Vinters,et al.  Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. , 1987, Science.

[34]  C. Barbas,et al.  Building zinc fingers by selection: toward a therapeutic application. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[35]  E. Poeschla,et al.  A controlled, Phase 1 clinical trial to evaluate the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a ribozyme that cleaves HIV-1 RNA. , 1998, Human gene therapy.

[36]  B. Korber,et al.  HIV sequence compendium 2002 , 2002 .

[37]  R. Morgan,et al.  Targeted transduction of CD34+ cells by transdominant negative Rev-expressing retrovirus yields partial anti-HIV protection of progeny macrophages. , 1998, Human Gene Therapy.

[38]  C. Barbas,et al.  Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[39]  D J Segal,et al.  Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins. , 2001, Current opinion in biotechnology.

[40]  D J Segal,et al.  Development of Zinc Finger Domains for Recognition of the 5′-ANN-3′ Family of DNA Sequences and Their Use in the Construction of Artificial Transcription Factors* , 2001, The Journal of Biological Chemistry.

[41]  B. Peterlin,et al.  Tat transactivation: a model for the regulation of eukaryotic transcriptional elongation. , 1999, Virology.

[42]  Aaron Klug,et al.  Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Ali Ehsani,et al.  Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells , 2002, Nature Biotechnology.

[44]  C. Pabo,et al.  Transcriptional Repression by Zinc Finger Peptides , 1997, The Journal of Biological Chemistry.

[45]  Jun Ma,et al.  GAL4-VP16 is an unusually potent transcriptional activator , 1988, Nature.

[46]  C. Barbas,et al.  Positive and negative regulation of endogenous genes by designed transcription factors. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[47]  H. Buc,et al.  HIV-1 reverse transcription. A termination step at the center of the genome. , 1994, Journal of molecular biology.

[48]  M. Churchill,et al.  A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. , 2000, Nucleic acids research.

[49]  H. Thiesen,et al.  Krüppel-associated boxes are potent transcriptional repression domains. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[50]  B. Ramratnam,et al.  Human Immunodeficiency Virus Type 1 Escape from RNA Interference , 2003, Journal of Virology.

[51]  C C Case,et al.  Regulation of an Endogenous Locus Using a Panel of Designed Zinc Finger Proteins Targeted to Accessible Chromatin Regions , 2001, The Journal of Biological Chemistry.

[52]  S. Vashee,et al.  Synergistic activation of transcription by physiologically unrelated transcription factors through cooperative DNA-binding. , 1998, Biochemical and biophysical research communications.

[53]  R. Eisenman,et al.  Mad proteins contain a dominant transcription repression domain , 1996, Molecular and cellular biology.

[54]  W. Gerlach,et al.  A phase I trial of autologous CD34+ hematopoietic progenitor cells transduced with an anti-HIV ribozyme. , 1999, Human gene therapy.

[55]  M. Reitz,et al.  Growth of macrophage-tropic and primary human immunodeficiency virus type 1 (HIV-1) isolates in a unique CD4+ T-cell clone (PM1): failure to downregulate CD4 and to interfere with cell-line-tropic HIV-1 , 1995, Journal of virology.