Immature dendritic cells (DCs) capture HIV type 1 (HIV-1) and can transmit captured virus particles to T cells. In this report, we show that HIV-1 particles captured by DCs can be transmitted to T cells by exocytosis without de novo infection. Captured HIV-1 particles were rapidly endocytosed to tetraspan protein (CD9, CD63)-positive endocytic compartments that were reminiscent of multivesicular endosomal bodies. Furthermore, some of the endocytosed virus particles were constitutively released into the extracellular milieu in association with HLA-DR1 ⁺ , CD1b ⁺ , CD9 ⁺ , and CD63 ⁺ vesicles (exosomes) and could initiate productive infections of CD4 ⁺ target cells. Surprisingly, the exocytosed vesicle-associated HIV-1 particles from DCs were 10-fold more infectious on a perparticle basis than cell-free virus particles. These studies describe a previously undescribed mechanism of DC-mediated HIV-1 transmission and suggest that virus particle trafficking to multivesicular endosomal bodies and subsequent exocytosis can provide HIV-1 particles captured by DCs an avenue for immune escape.

Tumor necrosis factor alpha (TNF-alpha) is a candidate human immunodeficiency virus type 1-induced neurotoxin that contributes to the pathogenesis of AIDS dementia complex. We report here on the effects of exogenous TNF-alpha on SK-N-MC human neuroblastoma cells differentiated to a neuronal phenotype with retinoic acid, TNF-alpha caused a dose-dependent loss of viability and a corresponding increase in apoptosis in differentiated SK-N-MC cells but not in undifferentiated cultures. Importantly, intracellular signalling via TNF receptors, as measured by activation of the transcription factor NF-kappa B, was unaltered by retinoic acid treatment. Finally, overexpression of bcl-2 or crmA conferred resistance to apoptosis mediated by TNF-alpha, as did the addition of the antioxidant N-acetylcysteine. These results suggest that TNF-alpha induces apoptosis in neuronal cells by a pathway that involves formation of reactive oxygen intermediates and which can be blocked by specific genetic interventions.

Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4+ T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81+ compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.

Human immunodeficiency virus type 1 (HIV-1) interactions with myeloid dendritic cells (DCs) can result in virus dissemination to CD4⁺ T cells via a trans infection pathway dependent on virion incorporation of the host cell derived glycosphingolipid (GSL), GM3. The mechanism of DC-mediated trans infection is extremely efficacious and can result in infection of multiple CD4⁺ T cells as these cells make exploratory contacts on the DC surface. While it has long been appreciated that activation of DCs with ligands that induce type I IFN signaling pathway dramatically enhances DC-mediated T cell trans infection, the mechanism by which this occurs has remained unclear until now. Here, we demonstrate that the type I IFN-inducible Siglec-1, CD169, is the DC receptor that captures HIV in a GM3-dependent manner. Selective downregulation of CD169 expression, neutralizing CD169 function, or depletion of GSLs from virions, abrogated DC-mediated HIV-1 capture and trans infection, while exogenous expression of CD169 in receptor-naïve cells rescued GSL-dependent capture and trans infection. HIV-1 particles co-localized with CD169 on DC surface immediately following capture and subsequently within non-lysosomal compartments that redistributed to the DC – T cell infectious synapses upon initiation of T cell contact. Together, these findings describe a novel mechanism of pathogen parasitization of host encoded cellular recognition machinery (GM3 – CD169 interaction) for DC-dependent HIV dissemination. Dendritic cells (DCs) are one of the initial cellular targets of HIV-1 and can play a crucial role in determining the course of virus infection in vivo. While sentinel functions of DCs are essential for establishment of an antiviral state, HIV-1 can subvert DC function for its dissemination. One of the mechanisms by which DCs can mediate virus spread is via the trans infection pathway whereby DCs capture HIV-1 particles and retain them in an infectious state without getting infected, and pass these infectious particles to CD4⁺ T cells upon initiation of cellular contacts. In this report, we demonstrate that expression of Siglec-1or CD169, on DC surface is responsible for capture of HIV-1 particles by binding the ganglioside, GM3, present in the virion lipid bilayer. This interaction between CD169 and GM3 targets captured virus particles to non-degradative compartments and resulted in retention of virus particle infectivity within DCs. Upon initiation of T cell contacts with virus-laden DCs, HIV-1 particles were trafficked to the DC – T synaptic junctions and transferred to T cells for establishment of productive infection. These studies define a novel host-encoded receptor – ligand interaction that drives HIV-1 dissemination and can be used for development of novel anti-viral therapeutics.

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70–Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70–Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70–Bag3 complex may offer an appealing anticancer target. Cancer Res; 74(17); 4731–40. ©2014 AACR.

The APOBEC3 family of mammalian cytidine deaminases, including APOBEC3G (A3G), has been shown to function as innate antiviral factors against retroviruses and can also suppress the replication of the hepatitis B virus (HBV). The mechanism by which A3G inhibits HBV replication remains to be elucidated. In this study, we show that the inhibitory effect of APOBEC3 proteins on HBV replication was mainly at the DNA level, with only a minor effect on viral RNA packaging. The anti-HBV effect of A3G was independent of the DNA-editing function, and the mode of inhibition was not due to HBV DNA degradation. The editing-independent antiviral activity of A3G could target DNA-RNA hybrids as well as single-stranded DNA. Finally, we show that there was a preferential decrease in the accumulation of longer minus-strand DNA by A3G, compared to the shorter minus-strand DNA, and suggest that A3G exerts its inhibitory effect at very early stages during viral reverse transcription.