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.

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.

Low levels of type I interferon (IFN-I) are thought to be a driving force for immune activation and T-cell exhaustion in HIV-1 infected individuals on combination antiretroviral therapy (cART), though the causative mechanisms for persistent IFN-I signaling have remained unclear. Here, we show Rev–CRM1-dependent nuclear export and peripheral membrane association of intron-containing HIV-1 RNA, independent of primary viral sequence or viral protein expression, is subject to sensing and signaling via MAVS, resulting in IFN-I-dependent pro-inflammatory responses in macrophages. Additionally, HIV-1 intron-containing-RNA-induced innate immune activation of macrophages leads to upregulation of inhibitory receptor expression and functional immune exhaustion of co-cultured T cells. Our findings suggest that persistent expression of HIV-1 intron-containing RNA in macrophages contributes to chronic immune activation and T-cell dysfunction and that use of HIV RNA expression inhibitors as adjunct therapy might abrogate aberrant inflammation and restore immune function in HIV-infected individuals on cART.

Protease inhibitors (PI) act by blocking human immunodeficiency virus (HIV) polyprotein processing, but there is no direct quantitative correlation between the degree of impairment of Gag processing and virion infectivity at low PI concentrations. To analyze the consequences of partial processing, virus particles were produced in the presence of limiting PI concentrations or by co-transfection of wild-type proviral plasmids with constructs carrying mutations in one or more cleavage sites. Low PI concentrations caused subtle changes in polyprotein processing associated with a pronounced reduction of particle infectivity. Dissection of individual stages of viral entry indicated a block in accumulation of reverse transcriptase products, whereas virus entry, enzymatic reverse transcriptase activity, and replication steps following reverse transcription were not affected. Co-expression of low amounts of partially processed forms of Gag together with wild-type HIV generally exerted a trans-dominant effect, which was most prominent for a construct carrying mutations at both cleavage sites flanking the CA domain. Interestingly, co-expression of low amounts of Gag mutated at the CA-SP1 cleavage site also affected processing activity at this site in the wild-type virus. The results indicate that low amounts (<5%) of Gag processing intermediates can display a trans-dominant effect on HIV particle maturation, with the maturation cleavage between CA and SP1 being of particular importance. These effects are likely to be important for the strong activity of PI at concentrations achieved in vivo and also bear relevance for the mechanism of action of the antiviral drug bevirimat.

Myeloid dendritic cells (DCs) can capture HIV-1 via the receptor CD169/Siglec-1 that binds to the ganglioside, GM3, in the virus particle membrane. In turn, HIV-1 particles captured by CD169, an I-type lectin, whose expression on DCs is enhanced upon maturation with LPS, are protected from degradation in CD169⁺ virus-containing compartments (VCCs) and disseminated to CD4⁺ T cells, a mechanism of DC-mediated HIV-1 trans-infection. In this study, we describe the mechanism of VCC formation and its role in immune evasion mechanisms of HIV-1. We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells. Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection. Furthermore, super-resolution microscopy revealed close association of CD169 and HIV-1 particles in surface-accessible but deep plasma membrane invaginations. Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization. Our study suggests that HIV-1 capture by CD169 can provide virus evasion from both innate (phagocytosis) and adaptive immune responses. Dendritic cells (DCs) are professional antigen presenting cells, and their sentinel roles are important to elicit a potent antiviral immunity. However, HIV-1 has exploited DCs to spread infection by several mechanisms. One such mechanism is the DC-mediated trans-infection pathway, whereby DCs transmit captured virus to CD4⁺ T cells. We have recently identified the type I interferon (IFN-I) inducible protein, CD169, as a receptor on DCs which mediates HIV-1 capture and trans-infection. We have also demonstrated extensive co-localization of HIV-1 with CD169 within peripheral non-lysosomal compartments in DCs, although the mechanism and biological importance of the compartment formation remain unclear. Here in this study, we report that a myeloid cell specific co-factor interacts with CD169 following virus capture leading to compartment formation. This co-factor is induced in DCs by an IFN-I-inducing TLR ligand LPS, but not by IFN-I itself. Though the CD169⁺ HIV-1 containing compartments are surface-accessible, these compartments have considerable depth and are connected to the surface, such that captured virus particles localized within these unique structures are protected from detection by anti-gp120 broadly neutralizing antibodies. Our study suggests that CD169–HIV-1 interaction provides an evasion mechanism from degradation by phagocytosis and neutralization by anti-viral humoral responses.

HIV-1 acquisition occurs most commonly after sexual contact. To establish infection, HIV-1 must infect cells that support high-level replication, namely CD4⁺ T cells, which are absent from the outermost genital epithelium. Dendritic cells (DCs), present in mucosal epithelia, potentially facilitate HIV-1 acquisition. We show that vaginal epithelial DCs, termed CD1a⁺ VEDCs, are unlike other blood- and tissue-derived DCs because they express langerin but not DC-SIGN, and unlike skin-based langerin⁺ DC subset Langerhans cells (LCs), they do not harbor Birbeck granules. Individuals primarily acquire HIV-1 that utilizes the CCR5 receptor (termed either R5 or R5X4) during heterosexual transmission, and the mechanism for the block against variants that only use the CXCR4 receptor (classified as X4) remains unclear. We show that X4 as compared with R5 HIV-1 shows limited to no replication in CD1a⁺ VEDCs. This differential replication occurs after fusion, suggesting that receptor usage influences postentry steps in the virus life cycle. Furthermore, CD1a⁺ VEDCs isolated from HIV-1–infected virologically suppressed women harbor HIV-1 DNA. Thus, CD1a⁺ VEDCs are potentially infected early during heterosexual transmission and also retain virus during treatment. Understanding the interplay between HIV-1 and CD1a⁺ VEDCs is important for future prevention and cure strategies.