Intranuclear Positions of HIV-1 Proviruses Are Dynamic and Do Not Correlate with Transcriptional Activity

The relationship between spatiotemporal distribution of HIV-1 proviruses and their transcriptional activity is not well understood. To elucidate the intranuclear positions of transcriptionally active HIV-1 proviruses, we utilized an RNA fluorescence in situ hybridization assay and RNA stem loops that bind to fluorescently labeled bacterial protein (Bgl-mCherry) to specifically detect HIV-1 transcription sites. Initially, transcriptionally active wild-type proviruses were located closer to the nuclear envelope (NE) than expected by random chance in HeLa (∼1.4 μm) and CEM-SS T cells (∼0.9 μm). Disrupting interactions between HIV-1 capsid and host cleavage and polyadenylation specificity factor (CPSF6) resulted in localization of proviruses to lamina-associated domains (LADs) adjacent to the NE in HeLa cells (∼0.9 - 1.0 μm); however, in CEM-SS T cells, there was little or no shift toward the NE (∼0.9 μm), indicating cell-type differences in the locations of transcriptionally active proviruses. The distance from the NE was not correlated with transcriptional activity, and transcriptionally active proviruses were randomly distributed throughout the HeLa cell after several cell divisions, indicating that the intranuclear locations of the chromosomal sites of integration are dynamic. After nuclear import HIV-1 cores colocalized with nuclear speckles, nuclear domains enriched in pre-mRNA splicing factors, but transcriptionally active proviruses detected 20 h after infection were mostly located outside but near nuclear speckles, suggesting a dynamic relationship between the speckles and integration sites. Overall, these studies establish that the nuclear distribution of HIV-1 proviruses is dynamic and the distance between HIV-1 proviruses and the NE does not correlate with transcriptional activity.