HIV-1 incorporation of host-cell–derived glycosphingolipid GM3 allows for capture by mature dendritic cells Wendy Blay Puryeara, Xinwei Yub, Nora P. Ramireza, Björn M. Reinhardb, and Suryaram Gummulurua,1 aDepartment of Microbiology, Boston University School of Medicine, Boston, MA 02118; and bDepartment of Chemistry and The Photonics Center, Boston University, Boston, MA 02215 Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved March 30, 2012 (received for review January 26, 2012) The interaction between HIV and dendritic cells (DCs) is an demonstrated that HIV-1 can bind to DCs in an Env-in- important early event in HIV-1 pathogenesis that leads to efficient dependent manner (10), and this mechanism of virus capture and viral dissemination. Here we demonstrate a HIV gp120-indepen- trans-infection is greatly enhanced upon maturation of DCs (11). dent DC capture mechanism that uses virion-incorporated host- HIV-1 is known to bud from specialized compartments of the derived gangliosides with terminal α2–3-linked sialic acid linkages. cell membrane known as membrane rafts (12). Membrane rafts Using exogenously enriched virus and artificial liposome particles, are defined as dynamic ordered assemblies with high levels of we demonstrate that both α2–3 gangliosides GM1 and GM3 are cholesterol, sphingomylein, glycosyphingolipids, and membrane capable of mediating this interaction when present in the particle proteins, such as tetraspanins and GPI-anchored proteins (13). at high levels. In the absence of overexpression, GM3 is the pri- Previous work in our laboratory has demonstrated a dramatic mary ligand responsible for this capture mechanism, because decrease in DC capture of HIV-1 that are derived from host cells siRNA depletion of GM3 but not GM1 from the producer cell and treated with inhibitors to the glycosphingolipid (GSL) biosynthesis hence virions, resulted in a dramatic decrease in DC capture. Fur- pathway (14, 15). As GSLs are a major component of membrane thermore, HIV-1 capture by DCs was competitively inhibited by rafts, it suggests that glycoprotein independent capture of HIV-1 targeting virion-associated GM3, but was unchanged by targeting by DCs is mediated by a host-derived glycosphingolipid, which is MICROBIOLOGY GM1. Finally, virions were derived from monocytoid THP-1 cells incorporated into the virion as it buds from host-cell plasma that constitutively display low levels of GM1 and GM3, or from membrane rafts. Here we demonstrate that GM3 is the host- THP-1 cells induced to express high surface levels of GM1 and GM3 derived GSL responsible for mediating the Env independent upon stimulation with the TLR2/1 ligand Pam3CSK4. Compared interaction between HIV-1 and mature DCs (mDCs). with untreated THP-1 cells, virus produced from Pam3CSK4-stimu- lated THP-1 cells incorporated higher levels of GM3, but not GM1, Results trans and showed enhanced DC capture and -infection. Our results Ganglioside Depletion Results in Virions That Are Deficient for mDC identify a unique HIV-1 DC attachment mechanism that is depen- Capture. GSLs make up approximately 5% of the overall mem- – dent on a host-cell derived ligand, GM3, and is a unique example brane lipid composition and are highly enriched in lipid rafts, of pathogen mimicry of host-cell recognition pathways that drive and, interestingly, enriched in HIV-1 particles as they bud from virus capture and dissemination in vivo. the host cell (16, 17). Our previous studies had suggested a re- quirement for GSLs in capture of both HIV Gag-eGFP virus-like f the estimated two and a half million new HIV infections particles (VLPs) and infectious HIV-1 particles (14, 15). This Othat occur each year (1), approximately 85% are acquired at requirement was observed with virus derived from HEK293T mucosal surfaces through sexual transmission (2). Dendritic cells cells, monocyte-derived macrophages (MDMs), and peripheral are postulated to be one of the first cells to contact virus, and are blood mononuclear cells (PBMCs), wherein GSL depletion in believed to play a central role in establishing infection and sub- the virus producer cell translated to virions with decreased mDC sequent viral dissemination (2). The majority of HIV/dendritic capture (Fig. S1). There are a large number of GSL variants, which cell (DC) interactions result in a virus that remains associated can be further classified as gangliosides, asialogangliosides, and with DCs in an infectious state (3). Captured virus particles can be globosides (Fig. S2). Gangliosides have a terminal sialic acid ’ + transferred to HIV-1 s primary target cell, the CD4 T cell (4) (NeuNAc) residue, whereas none of the other GSL subsets are through formation of infectious synapses (5), a process known as sialylated. To determine whether HIV-1 capture by mature DCs trans DC-mediated -infection (3). is dependent on sialylated GSLs, Gag-eGFP VLPs were treated As the only viral protein present on the surface of the virion, at 37 °C for >4 h with a neuraminidase (NA) that removes α2–3-, HIV-1 envelope (Env) provides the primary mechanism for α2–6-, and α2–8-linked NeuNAc from proteins and GSLs. NA- HIV-1 binding and entry. Although fusion of the virus particles treated or untreated VLPs were probed for the presence of VLP- to host-cell membranes require binding of HIV-1 Env to CD4 associated NeuNAc using an Alexa-594 conjugated wheat germ and coreceptor (CoR), several other Env attachment factors, agglutinin (WGA). The level of NeuNAc detected on NA-trea- fi such as dendritic cell-speci c intercellular adhesion molecule-3- ted VLPs was 2.8-fold lower than untreated VLPs (Fig. S3). The binding nonintegrin (3), syndecans (6), dendritic cell immuno- receptor (7), and galactosyl ceramide (8) are expressed by DCs and are believed to play key roles in binding and retention of Author contributions: W.B.P., B.M.R., and S.G. designed research; W.B.P., X.Y., and N.P.R. virus particles without fusion. Despite the central importance of performed research; X.Y. and B.M.R. contributed new reagents/analytic tools; W.B.P., Env in mediating HIV-1 binding and fusion, there are on average X.Y., B.M.R., and S.G. analyzed data; and W.B.P. and S.G. wrote the paper. only 7 to 14 irregularly spaced Env spikes on the surface of The authors declare no conflict of interest. a virion (9). This low Env density results in a virion surface that is This article is a PNAS Direct Submission. largely comprised of host-derived molecules and opens up the 1To whom correspondence should be addressed. E-mail: [email protected]. possibility that nonvirally encoded factors may also play impor- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tant roles in virus interactions with target cells. Indeed, we have 1073/pnas.1201104109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1201104109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 NA-treated VLPs show a marked deficit in capture, with a re- sphingolipid families, sphingomyelin and sulfatides, were depleted duction of 58% relative to untreated VLPs (Fig. 1A). This by knockdown of ceramide transport protein (CERT) and galac- finding suggests that the viral ligand responsible for glycoprotein tosyl transferase (UGT8), respectively. Knockdown of each of the independent mDC capture of VLPs is a ganglioside. targeted GSL biosynthetic enzymes was verified by RT-PCR and To further verify that ganglioside reduction results in decreased depletion of the targeted sphingolipids from the cell membrane – virus capture, we used siRNAs to create ganglioside-deficient was assessed by FACS analysis for cell-surface exposed GM1 (Fig. particles. HEK293T cells have a high level of endogenous gan- S4). Interestingly, a substantial decrease in VLP capture by mDCs gliosides and provided an ideal system for targeted siRNA was observed only with the virions produced from cells with knockdowns that impacted gangliosides (i.e., UGCG and knockdowns to produce ganglioside-deficient VLPs. Because gan- ST3Gal5), but not upon knockdowns of sphingomyelin (CERT) or gliosides are a subset of GSLs, the entire GSL family of sphingo- sulfatides (UGT8) (Fig. 1B). lipids was depleted from the cell by knockdown of glucosyl trans- To verify that the decreased mDC capture that we observed fi ferase (UGCG). This analysis was further re ned by targeting with ganglioside-depleted VLPs was recapitulated in the context GM3 synthase (ST3Gal5), which is responsible for all downstream of infectious virus, we performed a subset of the siRNA knock- ganglioside synthesis. As a comparison, the other two major downs described above and created replication competent HIVLai. The glycosyltransferases UGCG and ST3Gal5 were each targeted to produce ganglioside-depleted virus. Reduced incorporation of gangliosides in virus particles was verified by staining for the ganglioside GM3 and normalizing against the number of virions, determined by staining with an anti-p24gag antibody (representa- tive images and quantification shown in Fig. 1C, staining controls in Fig. S5). Because all other gangliosides are more complex derivations beyond GM3 (Fig. S2), the reduction of GM3 is a good indicator of generalized ganglioside depletion. In agreement with the VLP data, the ganglioside-depleted viruses were also signifi- cantly attenuated for capture by mDCs (>60% reduction) com- pared with HIVLai particles derived from nontargeting siRNA- transfected HEK293T cells (Fig. 1D). These results suggest that a virion-associated ganglioside plays a significant role in mDC capture of HIV-1 VLPs. Removal of Virion-Associated α2–3-Linked Sialic Acid Results in Virus Deficient for mDC Capture. Whereas all gangliosides possess a terminal NeuNAc, the number of residues and type of linkage varies. Relevant ganglioside linkages used in this study are in- dicated in Fig.