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Updated: Feb 11, 2026
Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform
Published on: May 18, 2021
Slow Release of HIV-1 Protein Nef from Vesicle-like Structures Is Inhibited by Cytosolic Calcium Elevation in Single
Matjaž Stenovec1,2, Eva Lasič2, Pia Pužar Dominkuš3
1Celica Biomedical, Tehnološki park 24, 1000, Ljubljana, Slovenia.
Abstract:
Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca2+ concentration ([Ca2+]i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P < 0.05), indicating that elevated [Ca2+]i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.
Insights
Microglia infected with HIV-1 release the accessory protein Nef via CD9 and CD81-positive compartments. Elevated intracellular calcium inhibits this Nef release, revealing a novel mechanism for HIV-1 regulation in microglia.
Area of Science:
- Neuroscience
- Virology
- Cell Biology
Background:
- Microglia, the immune cells of the brain, are primary targets of HIV-1 infection.
- The HIV-1 accessory protein Nef plays a crucial role in viral replication and pathogenesis.
- The intracellular trafficking and release mechanisms of Nef from microglia remain poorly understood.
Purpose of the Study:
- To investigate the intracellular compartmentalization, trafficking, and release of HIV-1 Nef protein from human microglia.
- To identify cellular compartments and molecular markers associated with Nef biogenesis and release.
Main Methods:
- Transfection of immortalized human microglia with Nef fused to green fluorescent protein (Nef.GFP).
- Biochemical fractionation and confocal microscopy to identify Nef.GFP localization and mobility.
- Analysis of the effect of intracellular calcium ([Ca2+]i) elevation on Nef.GFP structure mobility and release.
- Immunostaining for tetraspanins CD9 and CD81 to assess co-localization with Nef.GFP compartments.
Main Results:
- Nef.GFP localized to distinct vesicle-like structures, separate from conventional endo-/lysosomes.
- Nef.GFP structures exhibited slow, non-directional mobility, contrasting with faster, directional endosomal trafficking.
- Elevated intracellular calcium ([Ca2+]i) inhibited the release of Nef.GFP structures from microglia.
- Nef.GFP compartments significantly co-localized with CD9 and CD81 tetraspanins on plasma membrane-derived sites.
Conclusions:
- Microglial Nef protein is packaged into unique compartments distinct from classical endo-lysosomal pathways.
- The release of Nef from microglia is regulated by intracellular calcium levels.
- CD9 and CD81 tetraspanin-positive plasma membrane domains are involved in the biogenesis and release of microglial Nef.
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