BackgroundHuman immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages.MethodsMonocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1∆Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1∆Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines.ResultsHIV-1∆Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1β, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1∆Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1β and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1β and IL-8 in HIV-1wt more than in HIV-1∆Vpr-infected cultures. Supernatants from HIV-1∆Vpr-infected MDMs containing lower concentrations of IL-1β, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1β and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors.ConclusionCollectively, these results demonstrate the ability of HIV-1∆Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication.

BackgroundExtracellular vesicles (EVs) are nano-sized particles present in most body fluids including cerebrospinal fluid (CSF). Little is known about CSF EV proteins in HIV+ individuals. Here, we characterize the CSF EV proteome in HIV+ subjects and its relationship to neuroinflammation, stress responses, and HIV-associated neurocognitive disorders (HAND).MethodsCSF EVs isolated from 20 HIV+ subjects with (n = 10) or without (n = 10) cognitive impairment were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting, and untargeted LC/MS/MS mass spectrometry. Functional annotation was performed by gene ontology (GO) mapping and expression annotation using Biobase Transfac and PANTHER software. Cultured astrocytic U87 cells were treated with hydrogen peroxide for 4 h to induce oxidative stress and EVs isolated by ultracentrifugation. Selected markers of astrocytes (GFAP, GLUL), inflammation (CRP), and stress responses (PRDX2, PARK7, HSP70) were evaluated in EVs released by U87 cells following induction of oxidative stress and in CSF EVs from HIV+ patients by immunoblotting.ResultsMass spectrometry identified 2727 and 1626 proteins in EV fractions and EV-depleted CSF samples, respectively. CSF EV fractions were enriched with exosomal markers including Alix, syntenin, tetraspanins, and heat-shock proteins and a subset of neuronal, astrocyte, oligodendrocyte, and choroid plexus markers, in comparison to EV-depleted CSF. Proteins related to synapses, immune/inflammatory responses, stress responses, metabolic processes, mitochondrial functions, and blood-brain barrier were also identified in CSF EV fractions by GO mapping. HAND subjects had higher abundance of CSF EVs and proteins mapping to GO terms for synapses, glial cells, inflammation, and stress responses compared to those without HAND. GFAP, GLUL, CRP, PRDX2, PARK7, and HSP70 were confirmed by immunoblotting of CSF EVs from subjects with HAND and were also detected in EVs released by U87 cells under oxidative stress.ConclusionsThese findings suggest that CSF EVs derived from neurons, glial cells, and choroid plexus carry synaptic, immune/inflammation-related, and stress response proteins in HIV+ individuals with cognitive impairment, representing a valuable source for biomarker discovery.

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is transported through the secretory pathway to the infected cell surface and onto virion particles. In the Golgi, the gp160 Env precursor is modified by complex sugars and proteolytically cleaved to produce the mature functional Env trimer, which resists antibody neutralization. We observed mostly uncleaved gp160 and smaller amounts of cleaved gp120 and gp41 Envs on the surface of HIV-1-infected or Env-expressing cells; however, cleaved Envs were relatively enriched in virions and virus-like particles (VLPs). This relative enrichment of cleaved Env in VLPs was observed for wild-type Envs, for Envs lacking the cytoplasmic tail and for CD4-independent, conformationally flexible Envs. On the cell surface, we identified three distinct populations of Envs: 1) the cleaved Env was transported through the Golgi, was modified by complex glycans, formed trimers that crosslinked efficiently, and was recognized by broadly neutralizing antibodies; 2) a small fraction of Env modified by complex carbohydrates escaped cleavage in the Golgi; and 3) the larger population of uncleaved Env lacked complex carbohydrates, crosslinked into diverse oligomeric forms, and was recognized by poorly neutralizing antibodies. This last group of more "open" Env oligomers reached the cell surface in the presence of Brefeldin A, apparently bypassing the Golgi apparatus. Relative to Envs transported through the Golgi, these uncleaved Envs were counterselected for virion incorporation. By employing two pathways for Env transport to the surface of infected cells, HIV-1 can misdirect host antibody responses towards conformationally flexible, uncleaved Env without compromising virus infectivity. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The cleaved, functional Env is incorporated into virus particles from the surface of the infected cell. We found that an uncleaved form of Env is transported to the cell surface by an unconventional route, but this non-functional Env is mostly excluded from the virus. Thus, only one of the pathways by which Env is transported to the surface of infected cells results in efficient incorporation into virus particles, potentially allowing the uncleaved Env to act as a decoy to the host immune system without compromising virus infectivity.

Objective: The relationship of cerebrospinal fluid (CSF) extracellular vesicles to neurocognitive impairment (NCI) in HIV-infected individuals is unclear. Here, we characterize CSF extracellular vesicles and their association with central nervous system (CNS) injury related biomarkers [neurofilament light (NFL), S100B, neopterin] and NCI in HIV-positive individuals on combination antiretroviral therapy (cART). Design: A cross-sectional and longitudinal study of CSF samples from HIV-positive individuals on cART. Methods: NFL, S100B and neopterin were measured by ELISA in 190 CSF samples from 112 individuals (67 HIV-positive and 45 HIV-negative). CSF extracellular vesicles were isolated and characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting for exosome markers (CD9, CD63, CD81, FLOT-1) and ELISA for HLA-DR. Results: HIV-positive individuals had median age 52 years, 67% with suppressed plasma viral load (< 50 copies/ml), median CD4 + nadir 66 cells/μl and CD4 + cell count 313 cells/μl. CSF NFL, S100B and neopterin levels were higher in HIV-positive vs. HIV-negative individuals, and nonsuppressed vs. suppressed HIV-positive individuals. Although CSF NFL and S100B levels were higher in NCI vs. unimpaired HIV-positive individuals ( P < 0.05), only NFL was associated with NCI in adjusted models ( P < 0.05). CSF extracellular vesicles were increased in HIV-positive vs. HIV-negative individuals, and NCI vs. unimpaired HIV-positive individuals ( P < 0.05), and correlated positively with NFL ( P < 0.001). HLA-DR was enriched in CSF extracellular vesicles from HIV-positive individuals with NCI ( P < 0.05), suggesting that myeloid cells are a potential source of CSF extracellular vesicles during HIV infection. Conclusion: Increased CSF extracellular vesicles correlate with neuronal injury biomarker NFL in cART-treated HIV-positive individuals with neurocognitive impairment, suggesting potential applications as novel biomarkers of CNS injury.

Host immune components play both beneficial and pathogenic roles in human immunodeficiency virus type 1 (HIV-1) infection. During the initial stage of viral infection, a complex network of innate immune factors are activated. For instance, the immune cells express a number of inflammatory proteins including cytokines, chemokines, and antiviral restriction factors. These factors, specifically, interferons (IFNs) play a crucial role in antiviral defense system by modulating the downstream signaling events, by inducing maturation of dendritic cells (DCs), and by activation of macrophages, natural killer (NK) cells, and B and T cells. However, HIV-1 has evolved to utilize a number of strategies to overcome the antiviral effects of the host innate immune system. This review discusses the pathways and strategies utilized by HIV-1 to establish latent and persistent infection by defeating host's innate defense system.