No abstract

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/ human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/ HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/ migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4-5.2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.

Cell-to-cell communication is essen for the development of multicellular systems and is coordinated by soluble factors, exosomes, gap junction (GJ) channels, and the recently described tunneling nanotubes (TNTs). We and others have demonstrated that TNT-like structures are mostly present during pathogenic conditions, including HIV infection. However, the nature, function, and communication properties of TNTs are still poorly understood. In this manuscript, we demonstrate that TNTs induced by HIV infection have functional GJs at the ends of their membrane extensions and that TNTs mediate long-range GJ communication during HIV infection. Blocking or reducing GJ communication during HIV infection resulted in aberrant TNT cell-to-cell contact, compromising HIV spread and replication. Thus, TNTs and associated GJs are required for the efficient cell-to-cell communication and viral spread. Our data indicate that targeting TNTs/GJs may provide new therapeutic opportunities for the treatment of HIV.

Currently, a major barrier to curing HIV infection is the generation of tissue-associated, non-replicating, long-lasting viral reservoirs that are refractory to therapy and can be reactivated upon anti-retroviral therapy interruption. One of these reservoirs are latently HIV-infected macrophages. Here, we show that HIV infection of macrophages results in survival of a small population of infected cells that are metabolically altered and characterized by mitochondrial fusion, lipid accumulation, and reduced mitochondrial ATP production. No changes in glycolysis were detected. Metabolic analysis indicated an essential role of succinate and other TCA metabolites in the tricarboxylic acid (TCA) cycle in mediating lipid accumulation and oxidative phosphorylation (OXPHOS) in the mitochondria. Furthermore, we show that while uninfected and HIV infected macrophages use fatty acids and glucose as primary sources of energy, surviving HIV infected macrophages also use glutamine/glutamate as a major energy source, and blocking these new sources of energy resulted in the killing of latent HIV infected macrophages. Together, our data provide a new understanding of the formation, properties, and potential novel ways to eliminate macrophage viral reservoirs.

While HIV kills most of the cells it infects, a small number of infected cells survive and become latent viral reservoirs, posing a significant barrier to HIV eradication. However, the mechanism by which immune cells resist HIV-induced apoptosis is still incompletely understood. Here, we demonstrate that while acute HIV infection of human microglia/macrophages results in massive apoptosis, a small population of HIV-infected cells survive infection, silence viral replication, and can reactivate viral production upon specific treatments. We also found that HIV fusion inhibitors intended for use as antiretroviral therapies extended the survival of HIV-infected macrophages. Analysis of the pro- and anti-apoptotic pathways indicated no significant changes in Bcl-2, Mcl-1, Bak, Bax or caspase activation, suggesting that HIV blocks a very early step of apoptosis. Interestingly, Bim, a highly pro-apoptotic negative regulator of Bcl-2, was upregulated and recruited into the mitochondria in latently HIV-infected macrophages both in vitro and in vivo. Together, these results demonstrate that macrophages/microglia act as HIV reservoirs and utilize a novel mechanism to prevent HIV-induced apoptosis. Furthermore, they also suggest that Bim recruitment to mitochondria could be used as a biomarker of viral reservoirs in vivo.