Long-term HIV-1 infection of neural progenitor populations.

BACKGROUND: HIV can reside in the brain for many years. While astrocytes are known to tolerate long-term HIV infection, the potential of other neural cell types to harbour HIV is unclear. OBJECTIVE: To investigate whether HIV can persist in neural progenitor cell populations. DESIGN: A multipotent human neural stem cell line (HNSC.100) was used to compare HIV infection in neural progenitor and astrocyte cell populations. METHODS: Expression of cellular genes/proteins was analysed by real-time reverse transcriptase PCR, Western blot, immunocytochemistry and flow cytometry. Morphological properties of cells were measured by quantitative fluorescent image analysis. Virus release by cells exposed to HIV-1IIIB was monitored by enzyme-linked immunosorbent assay for Gag. Proviral copy numbers were determined by real-time PCR and early HIV transcripts by reverse transcriptase PCR. Rev activity was determined with a fluorescent-based reporter assay. RESULTS: Progenitor populations differed from astrocyte populations by showing much lower glial fibrillary acidic protein (GFAP) production, higher cell-surface expression of the CXCR4 chemokine receptor, higher Rev activity and distinct cell morphologies. HIV-exposed progenitor cultures released moderate amounts of virus for over 2 months and continued to display cell-associated HIV markers (proviral DNA, early HIV transcripts) during the entire observation period (115 days). Differentiation of HIV-infected progenitor cells to astrocytes was associated with transient activation of virus production. Long-term HIV infection of progenitor populations led to upregulation of GFAP and changes in cell morphology. CONCLUSION: These studies suggest that neural progenitor populations can contribute to the reservoir for HIV in the brain and undergo changes as a consequence of HIV persistence.

Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging.

The human immunodeficiency virus Rev protein is a post-transcriptional activator of HIV gene expression. Rev is a nucleocytoplasmic shuttle protein that displays characteristic nuclear/nucleolar subcellular localization in various cell lines. Cytoplasmic localization of Rev occurs under various conditions disrupting Rev function. The goal of this study was to investigate the relationship between localization of Rev and its functional activity in living cells. A triple-fluorescent imaging assay, called AQ-FIND, was established for automatic quantitative evaluation of nucleocytoplasmic distribution of fluorescently tagged proteins. This assay was used to screen 500 rev genes generated by error-prone PCR for Rev mutants with different localization phenotypes. Activities of the Rev mutants were determined with a second quantitative, dual-fluorescent reporter assay. In HeLa cells, the majority of nuclear Rev mutants had activities similar to wild-type Rev. The activities of Rev mutants with abnormal cytoplasmic localization ranged from moderately impaired to nonfunctional. There was no linear correlation between subcellular distribution and levels of Rev activity. In astrocytes, nuclear Rev mutants showed similar impaired activities as the cytoplasmic wild-type Rev. Our data suggest that steady-state subcellular localization is not a primary regulator of Rev activity but may change as a secondary consequence of altered Rev function. The methodologies described here have potential for studying the significance of subcellular localization for functions of other regulatory factors.