In a recent article published in the journal Molecular Cell, researchers present a valuable data resource that provides in-depth insights into cellular and genetic mechanisms governing human immunodeficiency virus (HIV) integration and expression in the human brain.
Study: HIV integration in the human brain is linked to microglial activation and 3D genome remodeling. Image Credit: SquareMotion / Shutterstock.com
HIV enters the central nervous system (CNS) within the first two weeks following infection. Microglia, myeloid cells, and macrophages, which collectively constitute 5-10% of neurons, are primarily infected.
HIV-infected with encephalitis (HIVE) is an uncommon complication following the discovery of combined antiretroviral therapy (cART); however, HIVE is the ideal condition for studying how active HIV replication impacts the brain. In some cases, during the initial stages of acute HIV infection, patients have neurological symptoms resembling a HIVE-like state.
More genome and transcriptome-level data are needed to design effective treatments for HIV-associated neurocognitive disorder (HAND), which affects between 20-50% of HIV-infected people globally. To date, most genomic studies have focused on gene expression profiling of bulk tissues to demonstrate HIV-triggered metabolic changes and neuroinflammation.
About the study
In the present study, researchers perform the first-of-its-kind cell-type-specific integrative genomics study encompassing three-dimensional (3D) genome mapping, HIV integration site sequencing (IS-seq), and single-nucleus transcription.
While prior studies used bulk tissues, the current study researchers used frontal lobe tissues from HIV-infected deceased donors and controls with and without encephalitis. More specifically, frontal cortex gray matter and subcortical white matter samples of three HIV-uninfected, three HIV-infected, and seven HIVE were obtained.
For the cell-type level resolution of transcription, the team performed 10X Chromium single-nucleus ribonucleic acid sequencing (snRNA-seq) on tissue samples from the brain's frontal lobe. Furthermore, the researchers screened single nuclei for HIV transcripts and observed HIV transcripts in all types of brain cells.
The 3D reorganization of microglia has not been visualized in vivo. Therefore, the team generated genome-wide scale high-throughput chromosome conformation capture (Hi-C) maps for microglia from two HIVE brains with age- and gender-matched controls. Per sc-RNA seq results, these HIVE brain samples had confirmed HIV expression.
Furthermore, the researchers examined 0.1 to 10 megabase (Mb)-sized chromosomal compartments of microglia cells that were subsequently divided into gene-dense and gene-poor A & B compartments. Notably, phase separation drives the spatial segregation of A and B chromosome compartments.
Since HIVE also remodels chromatin compartment architecture, the researchers explored topologically associated domains (TAD) and loop alterations in microglial cells.
Through snRNA-seq, 69,843 nuclei were profiled, which led to an average of 2,401 differentially expressed genes (DEGs) and a mean depth of 119,797 reads. In addition, differential compartment analysis of Hi-C (dcHiC) encompassing 194 Mb of the microglial genome, which is almost the size of human chromosome 5 in length, also revealed significant variations in A/B compartmentalization in the HIVE brain as compared to the HIV-infected brain.
While undergoing profound reorganization, chromosomal conformations at the site of DEGs in microglia also altered kilo- to mega-base-scaling TADs and rewired hundreds of contact-specific loops.
The genome in the A compartment in HIVE and HIV-infected microglia showed a marked shift from 47.3% to 46.2%. Additionally, the 118 Mb of chromosomal A/B-compartments of the HIVE microglia cells switched to a more open conformation, whereas 76 Mb transitioned toward a more closed conformation in the opposite direction.
Regions with augmented A compartmentalization in seven HIVE samples had 1,940 genes expressed at significantly higher levels than three HIV-infected microglia from the snRNA-seq dataset. Overall, Hi-C compartment changes in HIVE were microglia-specific.
Functional pathway analysis revealed a substantial enrichment in complement cascade genes, interferon (IFN), and other cytokine signaling pathways, as well as myeloid chemotaxis and migration pathways. These changes pointed to a shift in the microglia's functionality from neuronal support functions to inflammation due to HIV integration in the brain.
Remarkably, non-encephalitic microglia from HIV-infected brains showed conserved changes in the expression of genes related to neuronal health, even in the absence of HIV integration. An early insult likely leads to HAND and associated neuronal dysfunction during HIV infection, despite no direct neuronal infection.
The current study demonstrated that 3D genome-wide alterations in HIV-infected brains were microglia-specific and resulted in significant nuclear transcriptome reprogramming. For example, gene loci in HIVE microglia, with an upregulated expression, shifted to a more favorable transcriptional and open chromatin state, thus indicating that HIV integration and transcription caused these immune changes in microglia.
In addition, HIV-derived transcripts impacted microglial chromosomal conformations via other mechanisms. HIV showed a robust predilection for integration into A compartment open chromatin. Indeed, some HIV integration sites likely depend on disease stages similar to T-cells, which have been reported in previous studies.
Other viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also rewire chromosomal organization at the odorant receptor gene. The researchers noted a link between immune stimulation and infection, suggesting brain microglial activation potentiates HIV infection somehow.
These microglia are sites of high levels of productive HIV transcription, which later disseminates from here into other susceptible brain cell populations. These small clonal populations of HIV-integration sites replicate and increase the population of CNS viruses.
Overall, the study data showing cell-type-specific differences in HIV integration sites could support the development of next-generation HIV cure therapies.
- Plaza-Jennings, A. L., Valada, A., O’Shea, C., et al. (2022). HIV integration in the human brain is linked to microglial activation and 3D genome remodeling. Molecular Cell. doi:10.1016/j.molcel.2022.11.016