Dopamine, Immunity, and Disease.

The neurotransmitter dopamine is a key factor in central nervous system (CNS) function, regulating many processes including reward, movement, and cognition. Dopamine also regulates critical functions in peripheral organs, such as blood pressure, renal activity, and intestinal motility. Beyond these functions, a growing body of evidence indicates that dopamine is an important immunoregulatory factor. Most types of immune cells express dopamine receptors and other dopaminergic proteins, and many immune cells take up, produce, store, and/or release dopamine, suggesting that dopaminergic immunomodulation is important for immune function. Targeting these pathways could be a promising avenue for the treatment of inflammation and disease, but despite increasing research in this area, data on the specific effects of dopamine on many immune cells and disease processes remain inconsistent and poorly understood. Therefore, this review integrates the current knowledge of the role of dopamine in immune cell function and inflammatory signaling across systems. We also discuss the current understanding of dopaminergic regulation of immune signaling in the CNS and peripheral tissues, highlighting the role of dopaminergic immunomodulation in diseases such as Parkinson's disease, several neuropsychiatric conditions, neurologic human immunodeficiency virus, inflammatory bowel disease, rheumatoid arthritis, and others. Careful consideration is given to the influence of experimental design on results, and we note a number of areas in need of further research. Overall, this review integrates our knowledge of dopaminergic immunology at the cellular, tissue, and disease level and prompts the development of therapeutics and strategies targeted toward ameliorating disease through dopaminergic regulation of immunity. SIGNIFICANCE STATEMENT: Canonically, dopamine is recognized as a neurotransmitter involved in the regulation of movement, cognition, and reward. However, dopamine also acts as an immune modulator in the central nervous system and periphery. This review comprehensively assesses the current knowledge of dopaminergic immunomodulation and the role of dopamine in disease pathogenesis at the cellular and tissue level. This will provide broad access to this information across fields, identify areas in need of further investigation, and drive the development of dopaminergic therapeutic strategies.

HIV, Tat and dopamine transmission.

Human Immunodeficiency Virus (HIV) is a progressive infection that targets the immune system, affecting more than 37 million people around the world. While combinatorial antiretroviral therapy (cART) has lowered mortality rates and improved quality of life in infected individuals, the prevalence of HIV associated neurocognitive disorders is increasing and HIV associated cognitive decline remains prevalent. Recent research has suggested that HIV accessory proteins may be involved in this decline, and several studies have indicated that the HIV protein transactivator of transcription (Tat) can disrupt normal neuronal and glial function. Specifically, data indicate that Tat may directly impact dopaminergic neurotransmission, by modulating the function of the dopamine transporter and specifically damaging dopamine-rich regions of the CNS. HIV infection of the CNS has long been associated with dopaminergic dysfunction, but the mechanisms remain undefined. The specific effect(s) of Tat on dopaminergic neurotransmission may be, at least partially, a mechanism by which HIV infection directly or indirectly induces dopaminergic dysfunction. Therefore, precisely defining the specific effects of Tat on the dopaminergic system will help to elucidate the mechanisms by which HIV infection of the CNS induces neuropsychiatric, neurocognitive and neurological disorders that involve dopaminergic neurotransmission. Further, this will provide a discussion of the experiments needed to further these investigations, and may help to identify or develop new therapeutic approaches for the prevention or treatment of these disorders in HIV-infected individuals.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between HIV infection and cocaine use disorder is likely bidirectional, with cocaine use directly impacting immune function while HIV infection alters addiction-related behavior. To better characterize the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilizes a humanized mouse model to investigate the outcomes of HIV-1 infection on cocaine-related behaviors in a conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection selectively impairs cocaine CPP extinction without effecting reinstatement or cocaine seeking under conflict. Behavioral alterations are accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes are observed in human cytokines, including HIV-induced reductions in human TNFα, and cocaine and HIV interactions on GM-CSF levels. Together these data provide new insights into the unique neurobehavioral outcomes of HIV infection and cocaine exposure and how they interact to effect immune responses.

Dopamine-driven Increase in IL-1ß in Myeloid Cells is Mediated by Differential Dopamine Receptor Expression and Exacerbated by HIV.

The catecholamine neurotransmitter dopamine is classically known for regulation of central nervous system (CNS) functions such as reward, movement, and cognition. Increasing evidence also indicates that dopamine regulates critical functions in peripheral organs and is an important immunoregulatory factor. We have previously shown that dopamine increases NF-κB activity, inflammasome activation, and the production of inflammatory cytokines such as IL-1ß in human macrophages. As myeloid lineage cells are central to the initiation and resolution of acute inflammatory responses, dopamine-mediated dysregulation of these functions could both impair the innate immune response and exacerbate chronic inflammation. However, the exact pathways by which dopamine drives myeloid inflammation are not well defined, and studies in both rodent and human systems indicate that dopamine can impact the production of inflammatory mediators through both D1-like dopamine receptors (DRD1, DRD5) and D2-like dopamine receptors (DRD2, DRD3, and DRD4). Therefore, we hypothesized that dopamine-mediated production of IL-1ß in myeloid cells is regulated by the ratio of different dopamine receptors that are activated. Our data in primary human monocyte-derived macrophages (hMDM) indicate that DRD1 expression is necessary for dopamine-mediated increases in IL-1ß, and that changes in the expression of DRD2 and other dopamine receptors can alter the magnitude of the dopamine-mediated increase in IL-1ß. Mature hMDM have a high D1-like to D2-like receptor ratio, which is different relative to monocytes and peripheral blood mononuclear cells (PBMCs). We further confirm in human microglia cell lines that a high ratio of D1-like to D2-like receptors promotes dopamine-induced increases in IL-1ß gene and protein expression using pharmacological inhibition or overexpression of dopamine receptors. RNA-sequencing of dopamine-treated microglia shows that genes encoding functions in IL-1ß signaling pathways, microglia activation, and neurotransmission increased with dopamine treatment. Finally, using HIV as an example of a chronic inflammatory disease that is substantively worsened by comorbid substance use disorders (SUDs) that impact dopaminergic signaling, we show increased effects of dopamine on inflammasome activation and IL-1ß in the presence of HIV in both human macrophages and microglia. These data suggest that use of addictive substances and dopamine-modulating therapeutics could dysregulate the innate inflammatory response and exacerbate chronic neuroimmunological conditions like HIV. Thus, a detailed understanding of dopamine-mediated changes in inflammation, in particular pathways regulating IL-1ß, will be critical to effectively tailor medication regimens.

Gene expression and chromatin conformation of microglia in virally suppressed people with HIV.

The presence of HIV in sequestered reservoirs is a central impediment to a functional cure, allowing HIV to persist despite life-long antiretroviral therapy (ART), and driving a variety of comorbid conditions. Our understanding of the latent HIV reservoir in the central nervous system is incomplete, because of difficulties in accessing human central nervous system tissues. Microglia contribute to HIV reservoirs, but the molecular phenotype of HIV-infected microglia is poorly understood. We leveraged the unique "Last Gift" rapid autopsy program, in which people with HIV are closely followed until days or even hours before death. Microglial populations were heterogeneous regarding their gene expression profiles but showed similar chromatin accessibility landscapes. Despite ART, we detected occasional microglia containing cell-associated HIV RNA and HIV DNA integrated into open regions of the host's genome (∼0.005%). Microglia with detectable HIV RNA showed an inflammatory phenotype. These results demonstrate a distinct myeloid cell reservoir in the brains of people with HIV despite suppressive ART. Strategies for curing HIV and neurocognitive impairment will need to consider the myeloid compartment to be successful.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between progressive HIV infection and cocaine use disorder is likely bidirectional, with cocaine use having direct effects on immune function while HIV infection can alter addiction-related behavior. To better characterized the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilized a humanized mouse model to investigate the outcomes of progressive HIV infection on cocaine-related behaviors in a cocaine conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection did not impact the formation of a cocaine CPP, but did result in resistance to extinction of the CPP. No effects of HIV on yohimbine-primed reinstatement or cocaine seeking under conflict were observed. These behavioral alterations were accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes were observed in both mouse and human markers. Among other targets, this included HIV-induced reductions in mouse IL-1α and G-CSF and human TNFα and cocaine-induced alterations in human TNFα and mouse GM-CSF such that cocaine exposure increases both cytokines only in the absence of HIV infection. Together these data provide new insights into the unique neurobehavioral processes underlying HIV infection and cocaine use disorders, and further how they interact to effect immune responses.

Characterization and function of the human macrophage dopaminergic system: implications for CNS disease and drug abuse.

BACKGROUND: Perivascular macrophages and microglia are critical to CNS function. Drugs of abuse increase extracellular dopamine in the CNS, exposing these cells to elevated levels of dopamine. In rodent macrophages and human T-cells, dopamine was shown to modulate cellular functions through activation of dopamine receptors and other dopaminergic proteins. The expression of these proteins and the effects of dopamine on human macrophage functions had not been studied. METHODS: To study dopaminergic gene expression, qRT-PCR was performed on mRNA from primary human monocyte derived macrophages (MDM). Expression and localization of dopaminergic proteins was examined by immunoblotting isolated plasma membrane, total membrane and cytosolic proteins from MDM. To characterize dopamine-mediated changes in cytokine production in basal and inflammatory conditions, macrophages were treated with different concentrations of dopamine in the presence or absence of LPS and cytokine production was assayed by ELISA. Statistical significance was determined using two-tailed Students' T-tests or Wilcoxen Signed Rank tests. RESULTS: These data show that MDM express mRNA for all five subtypes of dopamine receptors, and that dopamine receptors 3 and 4 are expressed on the plasma membrane. MDM also express mRNA for the dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC). DAT is expressed on the plasma membrane, VMAT2 on cellular membranes and TH and AADC are in the cytosol. Dopamine also alters macrophage cytokine production in both untreated and LPS-treated cells. Untreated macrophages show dopamine mediated increases IL-6 and CCL2. Macrophages treated with LPS show increased IL-6, CCL2, CXCL8 and IL-10 and decreased TNF-α. CONCLUSIONS: Monocyte derived macrophages express dopamine receptors and other dopaminergic proteins through which dopamine may modulate macrophage functions. Thus, increased CNS dopamine levels due to drug abuse may exacerbate the development of neurological diseases including Alzheimer's disease and HIV associated neurological disorders.

Dopamine and norepinephrine are embracing their immune side and so should we.

While the history of neuroimmunology is long, the explicit study of neuroimmune communication, and particularly the role of catecholamines in neuroimmunity, is still emerging. Recent studies have shown that catecholamines, norepinephrine, epinephrine, and dopamine, are central to multiple complex mechanisms regulating immune function. These studies show that catecholamines can be released from both the nervous system and directly from immune cells, mediating both autocrine and paracrine signaling. This commentary highlights the importance of catecholaminergic immunomodulation and discusses new considerations needed to study the role of catecholamines in immune homeostasis to best leverage their contribution to disease processes for the development of new therapeutic approaches.

Severity of neonatal influenza infection is driven by type I interferon and oxidative stress.

Neonates exhibit increased susceptibility to respiratory viral infections, attributed to inflammation at the developing pulmonary air-blood interface. IFN I are antiviral cytokines critical to control viral replication, but also promote inflammation. Previously, we established a neonatal murine influenza virus (IV) model, which demonstrates increased mortality. Here, we sought to determine the role of IFN I in this increased mortality. We found that three-day-old IFNAR-deficient mice are highly protected from IV-induced mortality. In addition, exposure to IFNß 24 h post IV infection accelerated death in WT neonatal animals but did not impact adult mortality. In contrast, IFN IIIs are protective to neonatal mice. IFNß induced an oxidative stress imbalance specifically in primary neonatal IV-infected pulmonary type II epithelial cells (TIIEC), not in adult TIIECs. Moreover, neonates did not have an infection-induced increase in antioxidants, including a key antioxidant, superoxide dismutase 3, as compared to adults. Importantly, antioxidant treatment rescued IV-infected neonatal mice, but had no impact on adult morbidity. We propose that IFN I exacerbate an oxidative stress imbalance in the neonate because of IFN I-induced pulmonary TIIEC ROS production coupled with developmentally regulated, defective antioxidant production in response to IV infection. This age-specific imbalance contributes to mortality after respiratory infections in this vulnerable population.

Aging alters antiviral signaling pathways resulting in functional impairment in innate immunity in response to pattern recognition receptor agonists.

The progressive impairment of immunity to pathogens and vaccines with aging is a significant public health problem as the world population shifts to an increased percentage of older adults (> 65). We have previously demonstrated that cells obtained from older volunteers have delayed and defective induction of type I interferons and T cell and B cell helper cytokines in response to TLR ligands when compared to those from adult subjects. However, the underlying intracellular mechanisms are not well described. Herein, we studied two critical pathways important in the production of type I interferon (IFN), the interferon response factor 7 (pIRF7), and TANK-binding kinase (pTBK-1). We show a decrease in pIRF7 and pTBK-1 in cross-priming dendritic cells (cDC1s), CD4+ T cell priming DCs (cDC2s), and CD14dimCD16+ vascular patrolling monocytes from older adults (n = 11) following stimulation with pathway-specific agonists in comparison with young individuals (n = 11). The decrease in these key antiviral pathway proteins correlates with decreased phagocytosis, suggesting impaired function in Overall, our findings describe molecular mechanisms which explain the innate functional impairment in older adults and thus could inform us of novel approaches to restore these defects.

Functional characterization of the biogenic amine transporters on human macrophages.

Monocyte-derived macrophages (MDMs) are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remain nebulous. In the CNS, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine transporter (NET) and dopamine transporter (DAT) on human MDMs. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured MDMs, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immunomodulatory mechanism in response to LPS. LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the proinflammatory response to LPS. Our data introduce a potential role for DAT in the regulation of innate immunity.

Neurokinin-1 receptor signaling induces a pro-inflammatory transcriptomic profile in CD16+ monocytes.

Neurokinin-1 receptor (NK1R) signaling can be immunomodulatory and it can lead to preferential transmigration of CD14+CD16+ monocytes across the blood brain barrier, potentially promoting the development of inflammatory neurological diseases, such as neuroHIV. To evaluate how NK1R signaling alters monocyte biology, RNA sequencing was used to define NK1R-mediated transcriptional changes in different monocyte subsets. The data show that NK1R activation induces a greater number of changes in CD14+CD16+ monocytes (152 differentially expressed genes), than in CD14+CD16- monocytes (36 genes), including increases in the expression of NF-κB and components of the NLRP3 inflammasome pathway. These results suggest that NK1R may alter the inflammatory state of CD14+CD16+ monocytes, influencing the development of neuroinflammation.

Dopamine Levels Induced by Substance Abuse Alter Efficacy of Maraviroc and Expression of CCR5 Conformations on Myeloid Cells: Implications for NeuroHIV.

Despite widespread use of antiretroviral therapy (ART), HIV remains a major public health issue. Even with effective ART many infected individuals still suffer from the constellation of neurological symptoms now known as neuroHIV. These symptoms can be exacerbated by substance abuse, a common comorbidity among HIV-infected individuals. The mechanism(s) by which different types of drugs impact neuroHIV remains unclear, but all drugs of abuse increase central nervous system (CNS) dopamine and elevated dopamine increases HIV infection and inflammation in human myeloid cells including macrophages and microglia, the primary targets for HIV in the brain. Thus, drug-induced increases in CNS dopamine may be a common mechanism by which distinct addictive substances alter neuroHIV. Myeloid cells are generally infected by HIV strains that use the chemokine receptor CCR5 as a co-receptor, and our data indicate that in a subset of individuals, drug-induced levels of dopamine could interfere with the effectiveness of the CCR5 inhibitor Maraviroc. CCR5 can adopt distinct conformations that differentially regulate the efficiency of HIV entry and subsequent replication and using qPCR, flow cytometry, Western blotting and high content fluorescent imaging, we show that dopamine alters the expression of specific CCR5 conformations of CCR5 on the surface of human macrophages. These changes are not affected by association with lipid rafts, but do correlate with dopamine receptor gene expression levels, specifically higher levels of D1-like dopamine receptors. These data also demonstrate that dopamine increases HIV replication and alters CCR5 conformations in human microglia similarly to macrophages. These data support the importance of dopamine in the development of neuroHIV and indicate that dopamine signaling pathways should be examined as a target in antiretroviral therapies specifically tailored to HIV-infected drug abusers. Further, these studies show the potential immunomodulatory role of dopamine, suggesting changes in this neurotransmitter may also affect the progression of other diseases.

Human immunodeficiency virus (HIV) infection of human macrophages is increased by dopamine: a bridge between HIV-associated neurologic disorders and drug abuse.

The prevalence of human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) that result from HIV infection of the central nervous system is increasing. Macrophages, the primary target for HIV within the central nervous system, play a central role in HIV-induced neuropathogenesis. Drug abuse exacerbates HAND, but the mechanism(s) by which this increased neuropathology results in more severe forms of HAND in HIV-infected drug abusers is unclear. The addictive and reinforcing effects of many drugs of abuse, such as cocaine and methamphetamine, are mediated by increased extracellular dopamine in the brain. We propose a novel mechanism by which drugs of abuse intensify HIV neuropathogenesis through direct effects of the neurotransmitter dopamine on HIV infection of macrophages. We found that macrophages express dopamine receptors 1 and 2, and dopamine activates macrophages by increasing ERK 1 phosphorylation. Our results demonstrate for the first time that dopamine increases HIV replication in human macrophages and that the mechanism by which dopamine mediates this change is by increasing the total number of HIV-infected macrophages. This increase in HIV replication is mediated by activation of dopamine receptor 2. These findings suggest a common mechanism by which drugs of abuse enhance HIV replication in macrophages and indicate that the drug abuse-heightened levels of central nervous system dopamine could increase viral replication, thereby accelerating the development of HAND.

Methamphetamine Increases the Proportion of SIV-Infected Microglia/Macrophages, Alters Metabolic Pathways, and Elevates Cell Death Pathways: A Single-Cell Analysis.

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain.

Macrophage-derived simian immunodeficiency virus exhibits enhanced infectivity by comparison with T-cell-derived virus.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infect and productively replicate in macrophages and T lymphocytes. Here, we show that SIV virions derived from macrophages have higher levels of infectivity than those derived from T cells. The lower infectivity of T-cell-derived viruses is influenced by the quantity or type of mannose residues on the virion. Our results demonstrate that the cellular origin of a virus is a major factor in viral infectivity. Cell-type-specific factors in viral infectivity, and organ-specific or disease stage-specific differences in cellular derivation of virions, can be critical in the pathogenesis of HIV and AIDS.

Dopaminergic impact of cART and anti-depressants on HIV neuropathogenesis in older adults.

The success of combination antiretroviral therapy (cART) has transformed HIV infection into a chronic condition, resulting in an increase in the number of older, cART-treated adults living with HIV. This has increased the incidence of age-related, non-AIDS comorbidities in this population. One of the most common comorbidities is depression, which is also associated with cognitive impairment and a number of neuropathologies. In older people living with HIV, treating these overlapping disorders is complex, often creating pill burden or adverse drug-drug interactions that can exacerbate these neurologic disorders. Depression, NeuroHIV and many of the neuropsychiatric therapeutics used to treat them impact the dopaminergic system, suggesting that dopaminergic dysfunction may be a common factor in the development of these disorders. Further, changes in dopamine can influence the development of inflammation and the regulation of immune function, which are also implicated in the progression of NeuroHIV and depression. Little is known about the optimal clinical management of drug-drug interactions between cART drugs and antidepressants, particularly in regard to dopamine in older people living with HIV. This review will discuss those interactions, first examining the etiology of NeuroHIV and depression in older adults, then discussing the interrelated effects of dopamine and inflammation on these disorders, and finally reviewing the activity and interactions of cART drugs and antidepressants on each of these factors. Developing better strategies to manage these comorbidities is critical to the health of the aging, HIV-infected population, as the older population may be particularly vulnerable to drug-drug interactions affecting dopamine.

Dopamine Increases CD14+CD16+ Monocyte Transmigration across the Blood Brain Barrier: Implications for Substance Abuse and HIV Neuropathogenesis.

In human immunodeficiency virus-1 (HIV) infected individuals, substance abuse may accelerate the development and/or increase the severity of HIV associated neurocognitive disorders (HAND). It is proposed that CD14+CD16+ monocytes mediate HIV entry into the central nervous system (CNS) and that uninfected and infected CD14+CD16+ monocyte transmigration across the blood brain barrier (BBB) contributes to the establishment and propagation of CNS HIV viral reservoirs and chronic neuroinflammation, important factors in the development of HAND. The effects of substance abuse on the frequency of CD14+CD16+ monocytes in the peripheral circulation and on the entry of these cells into the CNS during HIV neuropathogenesis are not known. PBMC from HIV infected individuals were analyzed by flow cytometry and we demonstrate that the frequency of peripheral blood CD14+CD16+ monocytes in HIV infected substance abusers is increased when compared to those without active substance use. Since drug use elevates extracellular dopamine concentrations in the CNS, we examined the effects of dopamine on CD14+CD16+ monocyte transmigration across our in vitro model of the human BBB. The transmigration of this monocyte subpopulation is increased by dopamine and the dopamine receptor agonist, SKF 38393, implicating D1-like dopamine receptors in the increase in transmigration elicited by this neurotransmitter. Thus, elevated extracellular CNS dopamine may be a novel common mechanism by which active substance use increases uninfected and HIV infected CD14+CD16+ monocyte transmigration across the BBB. The influx of these cells into the CNS may increase viral seeding and neuroinflammation, contributing to the development of HIV associated neurocognitive impairments.

Development and characterization of positively selected brain-adapted SIV.

HIV is found in the brains of most infected individuals but only 30% develop neurological disease. Both viral and host factors are thought to contribute to the motor and cognitive disorders resulting from HIV infection. Here, using the SIV/rhesus monkey system, we characterize the salient characteristics of the virus from the brain of animals with neuropathological disorders. Nine unique molecular clones of SIV were derived from virus released by microglia cultured from the brains of two macaques with SIV encephalitis. Sequence analysis revealed a remarkably high level of similarity between their env and nef genes as well as their 3' LTR. As this genotype was found in the brains of two separate animals, and it encoded a set of distinct amino acid changes from the infecting virus, it demonstrates the convergent evolution of the virus to a unique brain-adapted genotype. This genotype was distinct from other macrophage-tropic and neurovirulent strains of SIV. Functional characterization of virus derived from representative clones showed a robust in vitro infection of 174xCEM cells, primary macrophages and primary microglia. The infectious phenotype of this virus is distinct from that shown by other strains of SIV, potentially reflecting the method by which the virus successfully infiltrates and infects the CNS. Positive in vivo selection of a brain-adapted strain of SIV resulted in a near-homogeneous strain of virus with distinct properties that may give clues to the viral basis of neuroAIDS.