Correlative Imaging to Detect Rare HIV Reservoirs and Associated Damage in Tissues.

Correlative light-electron microscopy (CLEM) has evolved in the last decades, especially after significant developments in sample preparation, imaging acquisition, software, spatial resolution, and equipment, including confocal, live-cell, super-resolution, and electron microscopy (scanning, transmission, focused ion beam, and cryo-electron microscopy). However, the recent evolution of different laser-related techniques, such as mass spectrometry imaging (MSI) and laser capture microdissection, could further expand spatial imaging capabilities into high-resolution OMIC approaches such as proteomic, lipidomics, small molecule, and drug discovery. Here, we will describe a protocol to integrate the detection of rare viral reservoirs with imaging mass spectrometry.

Astrocytes are HIV reservoirs in the brain: A cell type with poor HIV infectivity and replication but efficient cell-to-cell viral transfer.

The major barrier to eradicating Human immunodeficiency virus-1 (HIV) infection is the generation of tissue-associated quiescent long-lasting viral reservoirs refractory to therapy. Upon interruption of anti-retroviral therapy (ART), HIV replication can be reactivated. Within the brain, microglia/macrophages and a small population of astrocytes are infected with HIV. However, the role of astrocytes as a potential viral reservoir is becoming more recognized because of the improved detection and quantification of HIV viral reservoirs. In this report, we examined the infectivity of human primary astrocytes in vivo and in vitro, and their capacity to maintain HIV infection, become latently infected, be reactivated, and transfer new HIV virions into neighboring cells. Analysis of human brain tissue sections obtained from HIV-infected individuals under effective and prolonged ART indicates that a small population of astrocytes has integrated HIV-DNA. In vitro experiments using HIV-infected human primary astrocyte cultures confirmed a low percentage of astrocytes had integrated HIV-DNA, with poor to undetectable replication. Even in the absence of ART, long-term culture results in latency that could be transiently reactivated with histone deacetylase inhibitor, tumor necrosis factor-alpha (TNF-α), or methamphetamine. Reactivation resulted in poor viral production but efficient cell-to-cell viral transfer into cells that support high viral replication. Together, our data provide a new understanding of astrocytes' role as viral reservoirs within the central nervous system (CNS).

Role of Tunneling Nanotube-like Structures during the Early Events of HIV Infection: Novel Features of Tissue Compartmentalization and Mechanism of HIV Spread.

HIV has become a chronic disease despite the effective use of antiretroviral therapy (ART). However, the mechanisms of tissue colonization, viral evolution, generation of viral reservoirs, and compartmentalization are still a matter of debate due to the challenges involved in examining early events of infection at the cellular and molecular level. Thus, there is still an urgent need to explore these areas to develop effective HIV cure strategies. In this study, we describe the early events of tissue colonization and compartmentalization as well as the role of tunneling nanotube-like structures during viral spread in the presence and absence of effective antiretroviral treatment. To examine these mechanisms, NOD/SCID IL-2 RG-/- humanized mice were either directly infected with HIVADA or with low numbers of HIVADA-infected leukocytes to limit tissue colonization in the presence and absence of TAK779, an effective CCR5 blocker of HIV entry. We identify that viral seeding in tissues occurs early in a tissue- and cell type-specific manner (24-72 h). Reduction in systemic HIV replication by TAK779 treatment did not affect tissue seeding or spreading, despite reduced systemic viral replication. Tissue-associated HIV-infected cells had different properties than cells in the circulation because the virus continues to spread in tissues in a tunneling nanotube-like structure-dependent manner, despite ART. Thus, understanding these mechanisms can provide new approaches to enhance the efficacy of existing ART and HIV infection cure strategies.

HIV-1 Tat: Role in Bystander Toxicity.

HIV Tat protein is a critical protein that plays multiple roles in HIV pathogenesis. While its role as the transactivator of HIV transcription is well-established, other non-viral replication-associated functions have been described in several HIV-comorbidities even in the current antiretroviral therapy (ART) era. HIV Tat protein is produced and released into the extracellular space from cells with active HIV replication or from latently HIV-infected cells into neighboring uninfected cells even in the absence of active HIV replication and viral production due to effective ART. Neighboring uninfected and HIV-infected cells can take up the released Tat resulting in the upregulation of inflammatory genes and activation of pathways that leads to cytotoxicity observed in several comorbidities such as HIV associated neurocognitive disorder (HAND), HIV associated cardiovascular impairment, and accelerated aging. Thus, understanding how Tat modulates host and viral response is important in designing novel therapeutic approaches to target the chronic inflammatory effects of soluble viral proteins in HIV infection.

CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release.

Cellular ESCRT machinery plays pivotal role in HIV-1 budding and release. Extracellular stimuli that modulate HIV-1 egress are currently unknown. We found that CCL2 induced by HIV-1 clade B (HIV-1B) infection of macrophages enhanced virus production, while CCL2 immuno-depletion reversed this effect. Additionally, HIV-1 clade C (HIV-1C) was refractory to CCL2 levels. We show that CCL2-mediated increase in virus production requires Gag late motif LYPX present in HIV-1B, but absent in HIV-1C, and ALIX protein that recruits ESCRT III complex. CCL2 immuno-depletion sequestered ALIX to F-actin structures, while CCL2 addition mobilized it to cytoplasm facilitating Gag-ALIX binding. The LYPX motif improves virus replication and its absence renders the virus less fit. Interestingly, novel variants of HIV-1C with PYRE/PYKE tetrapeptide insertions in Gag-p6 conferred ALIX binding, CCL2-responsiveness and enhanced virus replication. These results, for the first time, indicate that CCL2 mediates ALIX mobilization from F-actin and enhances HIV-1 release and fitness.

A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation.

HIV-1 Tat protein contributes to HIV-neuropathogenesis in several ways including its ability to be taken up by uninfected bystander CNS cells and to activate inflammatory host genes causing synaptic injury. Here, we report that in the globally dominant HIV-1 clade C, Tat displays a naturally occurring polymorphism, R57S, in its basic domain, which mediates cellular uptake. We examined the effect of this polymorphism on Tat uptake and its consequences for cellular gene transactivation. In decapeptides corresponding to the basic domain, a R57S substitution caused up to a 70% reduction in uptake. We also used a transcellular Tat transactivation assay, where we expressed Tat proteins of HIV-1 clade B (Tat-B) or C (Tat-C) or their position 57 variants in HeLa cells. We quantified the secreted Tat proteins and measured their uptake by TZM-bl cells, which provide readout via an HIV-1 Tat-responsive luciferase gene. Transactivation by Tat-B was significantly reduced by R57S substitution, while that of Tat-C was enhanced by the reciprocal S57R substitution. Finally, we exposed microglia to Tat variants and found that R57 is required for maximal neuroinflammation. The R57S substitution dampened this response. Thus, genetic variations can modulate the ability of HIV-1 Tat to systemically disseminate neuroinflammation.

Cafeteria diet increases fat mass and chronically elevates lumbar sympathetic nerve activity in rats.

Obesity causes sympathetic activation that promotes atherosclerosis, end-organ damage, and hypertension. Because high-fat induced weight gain in rats elevates plasma leptin at 1 to 3 days after the onset of calorie-dense diets, we hypothesized that diet-induced overfeeding will increase sympathetic activity within 1 week after the onset of the regimen. To test this, we continuously measured sympathetic activity and blood pressure before and during the onset of diet-induced obesity using a high-calorie, cafeteria-style diet. Female Wistar rats, in which radiotelemeters had been implanted for continuous monitoring of lumbar sympathetic activity, mean arterial pressure, and heart rate, were randomly assigned to groups that received regular chow (control) or a cafeteria diet for a period of 15 days. This short-term, cafeteria-feeding regimen caused modest but nonsignificant increases in body weight (P=0.07) and a doubling of brown and white adipose tissue (P<0.01). The increases in fat mass were accompanied by elevations in plasma leptin (P<0.001) but no change in glucose. Overall heart rates and blood pressure were higher in cafeteria rats compared with controls (P<0.05). Cafeteria diet-induced weight gain caused increases in lumbar sympathetic nerve activity that became significant by the 12th day of the diet (P<0.001). These data show, for the first time, that the high-fat, cafeteria-style diet stimulates sustained increases in lumbar sympathetic neural drive in rats.