HIV-1 Particle Capture Research Articles

Comparative analysis of human microglial models for studies of HIV replication and pathogenesis

BackgroundHIV associated neurocognitive disorders cause significant morbidity and mortality despite the advent of highly active antiretroviral therapy. A deeper understanding of fundamental mechanisms underlying HIV infection and pathogenesis in the central nervous system is warranted. Microglia are resident myeloid cells of the brain that are readily infected by HIV and may constitute a CNS reservoir. We evaluated two microglial model cell lines (C20, HMC3) and two sources of primary cell-derived microglia (monocyte-derived microglia [MMG] and induced pluripotent stem cell-derived microglia [iPSC-MG]) as potential model systems for studying HIV-microglia interactions.ResultsAll four microglial model cells expressed typical myeloid markers with the exception of low or absent CD45 and CD11b expression by C20 and HMC3, and all four expressed the microglia-specific markers P2RY12 and TMEM119. Marked differences were observed upon gene expression profiling, however, indicating that MMG and iPSC-MG cluster closely together with primary human microglial cells, while C20 and HMC3 were similar to each other but very different from primary microglia. Expression of HIV-relevant genes also revealed important differences, with iPSC-MG and MMG expressing relevant genes at levels more closely resembling primary microglia. iPSC-MG and MMG were readily infected with R5-tropic HIV, while C20 and HMC3 lack CD4 and require pseudotyping for infection. Despite many similarities, HIV replication dynamics and HIV-1 particle capture by Siglec-1 differed markedly between the MMG and iPSC-MG.ConclusionsMMG and iPSC-MG appear to be viable microglial models that are susceptible to HIV infection and bear more similarities to authentic microglia than two transformed microglia cell lines. The observed differences in HIV replication and particle capture between MMG and iPSC-MG warrant further study.

SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1 Particles.

SERINC5 is a 10-transmembrane-domain cellular protein that is incorporated into budding HIV-1 particles and reduces HIV-1 infectivity by inhibiting virus-cell fusion. HIV-1 susceptibility to SERINC5 is determined by sequences in the viral Env glycoprotein gp120, and the antiviral effect of SERINC5 is counteracted by the viral accessory protein Nef. While the precise mechanism by which SERINC5 inhibits HIV-1 infectivity is unclear, previous studies have suggested that SERINC5 affects Env conformation. To define the effects of SERINC5 on Env conformation, we quantified the binding of HIV-1 particles to immobilized Env-specific monoclonal antibodies. We observed that SERINC5 reduced the binding of HIV-1 particles bearing a SERINC5-susceptible Env to antibodies that recognize the V3 loop, a soluble CD4 (sCD4)-induced epitope, and an N-linked glycan. In contrast, SERINC5 did not alter the capture of HIV-1 particles bearing the SERINC5-resistant Env protein. Moreover, the effect of SERINC5 on antibody-dependent virus capture was abrogated by Nef expression. Our results indicate that SERINC5 inhibits HIV-1 infectivity by altering the conformation of gp120 on virions and/or physical masking of specific HIV-1 Env epitopes.IMPORTANCE SERINC5 is a host cell protein that inhibits the infectivity of HIV-1 by a novel and poorly understood mechanism. Here, we provide evidence that the SERINC5 protein alters the conformation of the HIV-1 Env proteins and that this action is correlated with SERINC5's ability to inhibit HIV-1 infectivity. Defining the specific effects of SERINC5 on the HIV-1 glycoprotein conformation may be useful for designing new antiviral strategies targeting Env.

Transforming growth factor beta 1 up-regulates CD169 (sialoadhesin) expression on monocyte-derived dendritic cells: role in HIV sexual transmission.

Recent data describe CD169 (also called sialoadhesin or Siglec-1) as the main HIV-1 receptor expressed by mucosal dendritic cells involved in the capture of the virus and its transmission to target cells. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1), a cytokine found in abundance in semen, on the expression of CD169 on dendritic cells in order to characterize its potential role in the capture of HIV-1 particles by these antigen-presenting cells. Monocyte-derived dendritic cells (MDDCs) were cultured in the presence of lipopolysaccharide, pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α)] or different concentrations of TGF-β1, and analyzed for maturation marker and Siglec expression. The ability of MDDCs to capture HIV particles following the different treatments was also analyzed. TGF-β1 treatment promotes a significant increase of CD169 expression on MDDCs. This effect was specific since neither DC-SIGN nor other Siglec expressions were changed. The CD169 increase was due to a de-novo synthesis as evidenced by Western blot experiment. This up-regulation was well correlated to the concentration of TGF-β1 and associated with an increase of the MDDC ability to bind HIV particles. Interestingly, this phenomenon was independent of the maturation status of MDDCs. This study demonstrates that the most abundant cytokine present in semen (TGF-β1) is able to enhance specifically the expression of an important molecule (CD169) involved in the capture and transmission of HIV-1 particles from the mucosal lumen to the submucosal compartment. Our results suggest that this mechanism may play a relevant role in sexual HIV transmission.

The use of Nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells.

HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious virions from infected samples.

Inhibition of infection and transmission of HIV-1 and lack of significant impact on the vaginal commensal lactobacilli by carbohydrate-binding agents

A selection of carbohydrate-binding agents (CBAs) with different glycan specificities were evaluated for their inhibitory effect against HIV infection and transmission, and their interaction with vaginal commensal bacteria. Several assays were used for the antiviral evaluation: (i) cell-free virus infection of human CD4+ T lymphocyte C8166 cells; (ii) syncytium formation in co-cultures of persistently HIV-1-infected HUT-78/HIV-1 and non-infected CD4+ SupT1 cells; (iii) DC-SIGN-directed capture of HIV-1 particles; and (iv) transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ C8166 cells. CBAs were also examined for their interaction with vaginal commensal lactobacilli using several viability, proliferation and adhesion assays. The CBAs showed efficient inhibitory activity in the nanomolar to low-micromolar range against four events that play a crucial role in HIV-1 infection and transmission: cell-free virus infection, fusion between HIV-1-infected and non-infected cells, HIV-1 capture by DC-SIGN and transmission of DC-SIGN-captured virus to T cells. As candidate microbicides should not interfere with the normal human microbiota, we examined the effect of CBAs against Lactobacillus strains, including a variety of vaginal strains, a gastrointestinal strain and several non-human isolates. None of the CBAs included in our studies inhibited the growth of these bacteria in several media, affected their viability or had any significant impact on their adhesion to HeLa cell monolayers. The CBAs in this study were inhibitory to HIV-1 in several in vitro infection and transmission models, and may therefore qualify as potential microbicide candidates. The lack of significant impact on commensal vaginal lactobacilli is an important property of these CBAs in view of their potential microbicidal use.

Carbohydrate-Binding Agents Efficiently Prevent Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin (DC-SIGN)-Directed HIV-1 Transmission to T Lymphocytes

Exposure of HIV-1 to dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-expressing B-lymphoblast Raji cells (Raji/DC-SIGN) but not to wild-type Raji/0 cells results in the capture of HIV-1 particles to the cells as measured by the quantification of cell-associated p24 antigen. Cocultivation of HIV-1-captured Raji/DC-SIGN cells with uninfected CD4+ T lymphocyte C8166 cells results in abundant formation of syncytia within 36 h after cocultivation. Short preexposure of HIV-1 to carbohydrate-binding agents (CBA) dose dependently prevents the Raji/DC-SIGN cells from efficiently binding the virus particles, and no syncytia formation occurs upon subsequent cocultivation with C8166 cells. Thus, the mannose-specific [i.e., the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Narcissus pseudonarcissus agglutinin; and Cymbidium agglutinin (CA); the procaryotic cyanovirin-N (CV-N); and the monoclonal antibody 2G12) and N-acetylglucosamine-specific (i.e., the plant lectin Urtica dioica agglutinin) CBAs efficiently abrogate the DC-SIGN-directed HIV-1 capture and subsequent transmission to T lymphocytes. In this assay, the CD4-down-regulating cyclotriazodisulfonamide derivative, the CXCR4 and CCR5 coreceptor antagonists 1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl] - 1,4,8,11 - tetrazacyclotetradecane (AMD3100) and maraviroc, the gp41-binding enfuvirtide, and the polyanionic substances dextran sulfate (M(r) 5000), sulfated polyvinyl alcohol, and the naphthalene sulfonate polymer PRO-2000 were markedly less efficient or even completely ineffective. Similar observations were made in primary monocyte-derived dendritic cell cultures that were infected with HIV-1 particles that had been shortly pre-exposed to the CBAs CV-N, CA, HHA, and GNA and the polyanions DS-5000 and PRO-2000. The potential of CBAs, but not polyanions and other structural/functional classes of entry inhibitors, to impair DC-SIGN-expressing cells in their capacity of transmitting HIV to T lymphocytes might be an important property to be taken into consideration in the eventual choice to move microbicide candidate drugs to the clinical setting.