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A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation
Published on: September 19, 2010
In vitro modeling of HIV proviral activity in microglia
Lee A Campbell1, Christopher T Richie1, Yajun Zhang1
1Intramural Research Program, National Institute on Drug Abuse, Biomedical Research Center, Baltimore, MD, USA.
Abstract:
Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-κB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation.
Insights
Researchers developed a new microglia cell line, HIV-NanoLuc CHME-5, to study HIV infection in the brain. This model helps investigate HIV-associated neurocognitive disorders and proviral regulation.
Area of Science:
- Neuroscience
- Virology
- Genetics
Background:
- Microglia are key in HIV-associated neurocognitive disorders (HAND) pathogenesis due to productive HIV infection.
- HIV infection in microglia releases neurotoxic viral proteins and inflammatory compounds, impairing neuronal function.
- Existing models of brain HIV infection are limited, hindering research into HAND.
Purpose of the Study:
- To create a novel microglia cell line with an integrated HIV provirus.
- To establish a model that recreates hallmarks of HIV infection in the brain.
- To facilitate the study of HIV neuropathology and proviral regulation.
Main Methods:
- Utilized CRISPR/Cas9 gene editing to integrate a modified HIV provirus into CHME-5 microglia.
- Created HIV-NanoLuc CHME-5 cells where Gag-Pol is replaced with NanoLuciferase for LTR activity assay.
- Confirmed expression of NanoLuc and HIV protein Nef in the engineered cell line.
Main Results:
- HIV-NanoLuc CHME-5 cells confirmed to express NanoLuc and HIV Nef.
- Proviral activity was enhanced by pro-inflammatory factors (LPS, TNF-alpha) and Tat overexpression.
- CRISPR/Cas9 modification of TLR4 and NF-κB inhibition (sulfasalazine) reduced LPS-mediated proviral activation.
Conclusions:
- HIV-NanoLuc CHME-5 cells provide a valuable tool for studying HIV neuropathology.
- The model aids in understanding the regulation of HIV proviral activity in microglia.
- This research advances the study of HIV infection within the central nervous system.
