Virus-infected macrophages are the major therapeutic target in the brain tissue, even though the productive virus replication in these cells is necessary but not sufficient to cause HIV Associated Dementia (HAD). HAART is still generally recommended for patients with AIDS and in general this treatment has been effective in decreasing the incidence of HAD;however a significant group of patients remains refractory to HAART even when its CNS penetration is enhanced by supplementing drugs. For this reason alone, the activation of native cellular mechanisms inducing anti-viral resistance in cells migrating to various organs including CNS should be considered a viable alternative to add to the existing antiviral approaches for HAD. We have induced such responses mediated through soluble molecule [previously named HRF (HIV-1 Resistance Factor)] able to prevent virus replication in CD4+ T cells. Recently we identified the HRF as a truncated form of a potent transcription suppressor - CTCF (tCTCF). The biological activity of tCTCF was confirmed by several experimental methods: (1) the siRNA silencing of CTCF mRNA in HRF(+) cells reversed the HIV-1 resistance phenotype and reduced the biological activity of their cell culture supernatants;(2) depletion of tCTCF from HRF(+) cell culture supernatant abolished its antiviral activity;while (3) the affinity purified tCTCF induced antiviral activity in human macrophages;and (4) the affinity purified tCTCF inhibited the transcription of HIV-1 LTR promoted expression of reporter gene. Our preliminary data showed that upon HIV-1 exposure, CTCF from nuclear extracts of HRF(+) but not HRF(-) control cells bound to DNA probe comprising NF-:B, Sp1 and YB-1 binding sequences, possibly through its interaction with YB-1 protein. We hypothesize that since NF-:B and YB-1 binding sites are separated by only 6 nucleotides the CTCF/YB1 complex bound to the virus promoter might obstruct the formation of NF- :B/DNA binding, thus halting transcription of virus genome. Taken together we discovered a novel form of CTCF protein that mediates innate responses to HIV-1 in human macrophages. The main goal of this application is to prepare foundation for future delivery of tCTCF as a biotherapeutic for treatment of HIV-1 in CNS. In this grant application we propose: (1) To study the mechanism of tCTCF mediated cellular responses to HIV-1;(2) To determine the minimum tCTCF amino-acid sequence required for the induction of antiviral responses and efficacy assessment of tCTCF treatment in primary human macrophages;and (3) To determine the minimal CTCF toxicity and brain entry in vivo.

Public Health Relevance

Recently we identified the novel anti-HIV-1 protein called truncated form of a potent transcription suppressor - CTCF (tCTCF). tCTCF is a product of posttranslational processing of the full size CTCF protein and is secreted into extracellular space by HIV-1 resistant - tCTCF producing cells. The biological activity of tCTCF was confirmed by several experimental methods: (1) the siRNA silencing of full length CTCF mRNA in tCTCF producing cells reversed the HIV-1 resistance phenotype and reduced the biological activity of their cell culture supernatants;(2) depletion of tCTCF from tCTCF(+) cell culture supernatant abolished its antiviral activity;while (3) the affinity purified tCTCF induced antiviral activity in human macrophages;and (4) the affinity purified tCTCF inhibited the transcription of HIV-1 LTR promoted expression of reporter gene. Our preliminary data showed that upon HIV-1 exposure, CTCF from nuclear extracts of tCTCF producing but not control cells bound to DNA probe comprising NF-kB, Sp1 and YB-1 binding sequences, possibly through its interaction with YB-1 protein. We hypothesize that since NF-kB and YB-1 binding sites are separated by only 6 nucleotides the CTCF/YB1 complex bound to the virus promoter might obstruct the formation of NF-kB/DNA binding, thus halting transcription of virus genome. Taken together we discovered a novel form of CTCF protein that mediates innate responses to HIV-1 in human macrophages. The main goal of this application is to prepare foundation for future delivery of tCTCF as a biotherapeutic for treatment of HIV-1 in CNS.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS062649-03
Application #
7920156
Study Section
NeuroAIDS and other End-Organ Diseases Study Section (NAED)
Program Officer
Wong, May
Project Start
2008-09-30
Project End
2013-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
3
Fiscal Year
2010
Total Cost
$402,435
Indirect Cost
Name
St. Luke's-Roosevelt Institute for Health Sciences
Department
Type
DUNS #
623216371
City
New York
State
NY
Country
United States
Zip Code
10019
Sachdeva, Rakhee; Li, Yuchang; Shilpi, Rasheda Y et al. (2015) Human X-DING-CD4 mediates resistance to HIV-1 infection through novel paracrine-like signaling. FEBS J 282:937-50
Sachdeva, Rakhee; Shilpi, Rasheda Y; Simm, Malgorzata (2014) The interplay between the X-DING-CD4, IFN-* and IL-8 gene activity in quiescent and mitogen- or HIV-1-exposed PBMCs from HIV-1 elite controllers, AIDS progressors and HIV-negative controls. Innate Immun 20:173-83
Sachdeva, Rakhee; Darbinian, Nune; Khalili, Kamel et al. (2013) DING proteins from phylogenetically different species share high degrees of sequence and structure homology and block transcription of HIV-1 LTR promoter. PLoS One 8:e69623
Ivanova, Anna; Shilpi, Rasheda Y; Sachdeva, Rakhee et al. (2012) Native X-DING-CD4 protein secreted by HIV-1 resistant CD4+ T cells blocks activity of IL-8 promoter in human endothelial cells infected with enteric bacteria. Innate Immun 18:571-579
Shilpi, Rasheda Y; Sachdeva, Rakhee; Simm, Malgorzata (2012) Cellular resistance to HIV-1 infection in target cells coincides with a rapid induction of X-DING-CD4 mRNA: indication of the unique host innate response to virus regulated through function of the X-DING-CD4 gene. Innate Immun 18:563-70
Sachdeva, Rakhee; Simm, Malgorzata (2011) Application of linear polyacrylamide coprecipitation of denatured templates for PCR amplification of ultra-rapidly reannealing DNA. Biotechniques 50:217-9
Lesner, Adam; Shilpi, Rasheda; Ivanova, Anna et al. (2009) Identification of X-DING-CD4, a new member of human DING protein family that is secreted by HIV-1 resistant CD4(+) T cells and has anti-viral activity. Biochem Biophys Res Commun 389:284-9