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Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.

ABSTRACT: HIV-1 entry into CD4(+) T cells requires binding of the virus to CD4 followed by engagement of either the C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4) coreceptor. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. We have used zinc-finger nucleases to drive the simultaneous genetic modification of both ccr5 and cxcr4 in primary human CD4(+) T cells. These gene-modified cells proliferated normally and were resistant to both CCR5- and CXCR4-using HIV-1 in vitro. When introduced into a humanized mouse model of HIV-1 infection, these coreceptor negative cells engraft and traffic normally, and are protected from infection with CCR5- and CXCR4-using HIV-1 strains. These data suggest that simultaneous disruption of the HIV coreceptors may provide a useful approach for the long-term, drug-free treatment of established HIV-1 infections.

SUBMITTER: Didigu CA

PROVIDER: S-EPMC3879906 | biostudies-other | 2014 Jan

REPOSITORIES: biostudies-other

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Publications

Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.

Didigu Chuka A CA Wilen Craig B CB Wang Jianbin J Duong Jennifer J Secreto Anthony J AJ Danet-Desnoyers Gwenn A GA Riley James L JL Gregory Phillip D PD June Carl H CH Holmes Michael C MC Doms Robert W RW

Blood 20131025 1

HIV-1 entry into CD4(+) T cells requires binding of the virus to CD4 followed by engagement of either the C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4) coreceptor. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. We have used zinc-finger nucleases to drive the simultaneous genetic modification of both ccr5 and cxcr4 in primary human CD4(+) T cells. These gene-modified ...[more]

PMID: 24162716

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Project description:Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of human ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in tumour cell lines. The hypothesis tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two tumour cell lines (H460 and HCT116). All four lines had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p=0.002) and 4.3±0.8% (p=0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no effect on glycolysis as measured by glucose consumption or lactate formation under oxic or anoxic conditions, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis. Use Affymetrix microarrays to examine the effecs of knockout of the gene ADPGK using Zinc Finger nucleases in two cultured cell lines: (i) HCT116 cells (three wild type cultures compared to three ADPGK-knockout cultures), (ii) H460 cells (two wild type cultures compared to two ADPGK-knockout cultures). Ten microarrays in total.

Dataset Information

Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.

Publications

Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.

Similar Datasets

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