HIV-1 incorporation of host-cell–derived 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 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 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 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 (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 (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- families, and , 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 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). 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. S2. To further discern the type of ganglioside acting as a viral ligand, we treated virions with either an α2–3 NeuNAc-specific neuraminidase or a broadly acting α2–3, 2–6, 2–8 NeuNAc neuraminidase at 37 °C for >4 h. The HIV-1 gly- coprotein is heavily glycosylated and contains several NeuNAc, which could bias these results. We therefore performed these assays with HIVLai lacking glycoprotein (HIVLaiΔEnv). Virons treated with either of the two neuraminidases displayed a similar deficit in capture by mDCs (Fig. 1E), suggesting that of the many possible gangliosides that could be incorporated into the virion, the GSL(s) that possesses a terminal α2–3 NeuNAc linkage serves as the viral ligand necessary for capture by mDCs.

Enrichment of α2–3-Linked Gangliosides Results in Enhanced HIV-1 Capture by mDCs. Because depletion of α2–3-linked gangliosides from the virion results in particles that are deficient for mDC capture, we predicted that enrichment of the virion with α2–3- linked gangliosides would result in enhanced mDC capture. Our α – Fig. 1. Gangliosides with 2 3 NeuNAc linkages are important for HIV-1 analyses focused on GM3 and GM1, because both α2–3-linked capture by mDCs. (A) Gag-eGFP VLPs were mock treated or treated with gangliosides were previously reported to populate lipid rafts (13) 0.5 units/μL α2–3, 2–6, 2–8 NA. (B) Gag-eGFP VLPs were derived from siRNA- and HIV-1 particles (12, 18). We used three independent strat- treated HEK293T cells. NT, nontargeting; UGT8, galactosyl transferase; CERT, fi ceramide transfer protein; UGCG, glucosyltransferases, ST3, GM3 transferase. egies to test this hypothesis: exogenous addition of lipid, arti cial Capture of VLPs by mDCs was analyzed by FACS (A and B). Data are reported as liposomes, and ganglioside up-regulation during monocyte acti- percentage of eGFP+ mDCs normalized to NT-treated VLPs. (C and D) Gan- vation or differentiation into a macrophage. fi glioside-de cient HIVLai was derived from HEK293T cells knocked down for Replication competent HIVLai were created from HeLa cells, NT, UGCG, or ST3. (C) Virions were labeled for p24gag (green) and GM3 (red). which in contrast to HEK293T cells, have a low level of endog- Representative fields are shown and the average mean fluorescent intensity enous gangliosides. HeLa cells were transiently transfected with (MFI) of GM3 normalized to p24gag ± SD is reported, *P < 0.001, one-way ’ HIVLai-expressing plasmids and cultured in the presence of ex- ANOVA with Dunnett s multiple comparison. (D) Fold decrease of ganglioside- ogenous lipid, either GM3 or GM1, which integrated into the cell depleted HIVLai capture relative to NT-treated viruses by mDCs is reported. (E) Δ μ α – – membrane before virus budding. Lipid enrichment of the cell was Fold decrease in HIVLai Env virus capture treated with 0.5 units/ L 2 3, 2 6, fi A D 2–8NAorα2–3-specific NA relative to mock-treated viruses by mDCs is veri ed by FACS (Fig. 2 for GM3 and Fig. 2 for GM1). Direct reported. All capture assays represent averaged data from a minimum of three staining of the virus particles verified that the lipid enrichment of donors, ±SEM, one-sample t test, *P < 0.05, **P < 0.01, ***P < 0.001. the producer cell translated into a significant enrichment of the

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1201104109 Puryear et al. Downloaded by guest on September 25, 2021 membrane proteins in addition to the virally encoded glycopro- tein (17). To further verify the role of GM3 and GM1 in mediating HIV-1 capture by mDC, we used a system wherein liposomes were constructed to represent a very basic and simplified model of a virus particle. The lipid composition of the HIV-1 membrane has been pre- viously analyzed quantitatively by mass spectrometry and shown to comprise 45.1% (molar percentage) cholesterol, 8.8% phos- phatidyl choline (PC), 4.4% phosphatidylethanolamine (PE), 14.8% plasmalogen-PE (PI-PE), and 8.4% phosphatidylserine (PS) (16). We chose lipids with a simplified composition and comparable size to assemble liposomes that approximate the physicochemical properties of the virus membrane (Fig. 3A and Table S1). Liposomes were further given a fluorescent tag to enable ready detection by FACS analysis. These base-level lip- osomes comprising dipalmitoylphosphatidylcholine (DPPC), PS, and cholesterol are herein referred to as “blank” liposomes. We then created different versions of these liposomes by introducing an additional 1% of various phospholipids. In ad- dition to the α2–3-linked gangliosides GM3 and GM1, we also created liposomes using the core phospholipid ceramide (Cer), galactosyl ceramide (Gal), to represent alternative phospholipid pathways, and tetrasialoganglioside GQ1b (GQ1b) to represent an α2–8-linked ganglioside with a complex branching structure. Mature DCs were challenged with equal amounts of liposomes and the level of capture was assayed by FACS analysis. Both

the GM3 and GM1 liposomes were captured at a significantly MICROBIOLOGY

Fig. 2. Enrichment of HIV-1 particles with α2–3-linked gangliosides results

in enhanced capture by mDCs. HIVLai particles were derived from HeLa cells treated with MeOH (mock), exogenous GM3 (+GM3) (A–C), or exogenous GM1 (+GM1) (D–F). (A and D) Enriched GSLs were detected on the cell membrane using (A) α-GM3 mAb or (D) CtxB and analyzed by FACS. Histo- grams for mock (gray line) and soluble (A) +GM3 or (D) +GM1 (black line) HeLa cells are shown. Gray fill shows secondary Ab-only control for A and unstained control for D.(B and E) GSL enrichment of virions was analyzed by staining for p24gag (green) and (B) GM3 (red) or (E) GM1 (red). Represen- tative fields are shown and the average MFI normalized to p24gag ± SD is

reported, **P < 0.001, Student’s t test. (C and F) Capture of HIVLai enriched with (C) GM3 or (F) GM1 by mDCs was analyzed by detecting p24gag content in cell lysates by ELISA. Values represent the average from three donors and are reported as fold enhancement relative to mock-treated virus, ±SEM, *P < 0.05, **P < 0.001, one-sample t test.

α – B E Fig. 3. Liposomes with 2 3-linked gangliosides are captured by mDCs and ganglioside in virus particles (Fig. 2 for GM3 and Fig. 2 for compete HIV-1 for binding. Lipid vesicles comprised 54% DPPC, 1% PS, 45% GM1). There was a significant enhancement in capture of both cholesterol, and a fluorescent tag (blank). Modified vesicles contained 1% of GM3- and GM1-enriched virus particles by mDCs compared Cer, Gal, GM3 (α2–3 linked), GM1 (α2–3 linked), or GQ1b (α2–8 linked). (A) with virus derived from untreated virus producer cells (Fig. 2 C Representative transmission electron microscope negative stain of 1% GM3 fi and F, respectively). liposomes at 25k magni cation. (B) Liposomes were incubated with mDCs and analyzed for MFI by FACS. Data are normalized to blank liposomes and represent the average from at least three donors, ±SEM, *P < 0.001, one- Liposome Model of Virions Shows That the Inclusion of α2–3-Linked sample t test. (C and D) Competition of virus (C) or VLP (D) capture by mDCs Gangliosides Results in Enhanced mDC Capture and Can Compete for by increasing amounts of liposomes was analyzed by detecting p24gag con- + Virion Binding. The surface of an HIV-1 virion is complex, con- tent in cell lysates by ELISA (C) or eGFP cells by FACS (D). Data shown taining a myriad of host-derived sphingolipids, cholesterol, and represent the mean of three donors.

Puryear et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 enhanced level in comparison with blank liposomes or other derivatives (Fig. 3B). We next used the liposome panel to compete with virus cap- ture by mDCs. On the basis of the capture profiles, we predicted that only the GM3- and GM1-enriched liposomes would com- petitively inhibit virus capture by mDCs. HIV-1 Env is known to bind GM3 (19); therefore, HIVLai devoid of glycoprotein (HIVLaiΔEnv) and Gag-eGFP VLPs were used in these assays. Mature DCs were first treated with 0.1% sodium azide (NaN3)to prevent membrane recycling and internalization of the liposome. Congruent with the capture data, both GM3- and GM1-enriched liposomes, but not Cer-, Gal-, or GQ1b-enriched liposomes, substantially inhibited mDC capture of both HIVLaiΔEnv par- ticles and VLPs (Fig. 3 C and D). These results suggest that mDCs encode recognition machinery that specifically binds α2– 3-linked gangliosides such as GM3 and GM1 and that this mechanism is used to recognize HIV-1 particles even in the absence of Env.

Both GM1 and GM3 Are Up-Regulated upon Macrophage Differ- entiation, but only GM3 Shows Increased Virion Incorporation for Enhanced mDC Capture. Although the previous assays demon- strated that modifications in the level of particle-associated GM3 or GM1 impacts the level of mDC capture, we sought to further verify this effect in a physiologically relevant cell type with nat- urally occurring differences in ganglioside levels. Whereas mon- ocytes express a low level of membrane-associated GM3, this level is up-regulated either upon differentiation into macro- phages (20) or upon immune activation (21). This dichotomy creates a tractable system wherein viruses are predicted to pos- sess varying levels of GM3 when produced from differentially stimulated monocytes or macrophages. The monocytic cell line THP-1 can undergo macrophage differentiation upon stimula- tion with the TLR2/1 ligand Pam3CSK4 (21). We therefore used Fig. 4. HIV-1 derived from Pam3CSK4-stimulated monocytoid cells have THP-1 cells that were either left untreated (GSLlo) or Pam3CSK4 hi increased levels of GM3 and display enhanced capture by mDCs. (A and B) stimulated to induce macrophage differentiation (GSL )before Cell surface expression of GSLs on THP1 cells was determined using (A) the production of virus particles. α-GM3 mAb or (B) CtxB and analyzed by FACS. Histograms for untreated As expected, Pam3CSK4 stimulated cells showed higher levels (gray line) and Pam3CSK4-stimulated (black line) cells are shown. Gray fill of both GM3 (Fig. 4A) and GM1 (Fig. 4B) than the untreated cells shows secondary Ab-only control for A and unstained control for B.(C and D) (Fig. 4 A and B). In agreement with data from exogenously HIVLai/YU2 derived from untreated or Pam3CSK4-treated THP-1 cells were C F incubated with mDCs and (C) analyzed by p24gag ELISA for virus capture or enriched HIVLai particles (Fig. 2 and ), viruses derived from + hi (D) washed and cocultured with autologous CD4 T cells. Cocultures were Pam3CSK4-stimulated THP-1 cells (GSL ) were captured to a gag greater extent by mDCs than those derived from untreated THP-1 stained for cell surface expression of CD3 and intracellular p24 , and the number of dual-positive T cells was determined by FACS. Values were nor- cells (GSLlo) (Fig. 4C) and were transferred to T cells at an en- D malized to those observed with virus derived from untreated THP-1 cells and hanced level (Fig. 4 ). Furthermore, enhanced mDC capture of are reported as average fold increase from a minimum of four donors.

virus particles derived from Pam3CSK4-stimulated THP-1 cells ±SEM, *P < 0.05, **P < 0.01, one-sample t test. (E and F)HIVLai/YU2 derived was independent of the presence of exosomes in virus-containing from untreated (mock) or Pam3CSK4-stimulated (+Pam3CSK4) THP-1 cells supernatants (Fig. S6). were labeled for p24gag and (E) GM3 or (F) GM1. A minimum of 10 fields To verify that the observed cellular up-regulation in GM3 and were quantified for MFI of (E) GM3 or (F) GM1 normalized to that of p24gag; GM1 translated into virions with increased levels of these gan- average MFI ± SD is shown, *P < 0.0001, Student’s t test. gliosides, virus particles were stained for GM3 or GM1. Surpris- ingly, although virions produced from GSLhi THP-1 cells showed (B4Galt), which is responsible for complex gangliosides and a higher level of GM3 than those produced from GSLlo THP-1 asialogangliosides (Fig. S2). The B4Galt knockdown acts down- cells (Fig. 4E), the level of GM1 was equivalent (Fig. 4F). This suggests that even though both GM3 and GM1 are up-regulated stream of GM3, leaving GM3 levels unchanged, although causing upon monocyte activation and both GM3 and GM1 are capable of a decrease in GM1. As a comparison we also repeated the mediating mDC capture (Figs. 2 and 3), only GM3 is incorporated knockdown of the glucosyl transferase (UGCG), which impacts all into the virion at levels sufficient for this capture mechanism. , and GM3 synthase (ST3Gal5) to target all gangliosides. Whereas UGCG and ST3Gal5 knockdowns resulted Blocking GM3-Dependent Interactions Results in a Decrease in mDC in virions deficient for mDC capture, B4Galt knockdowns had no Capture of HIV-1 Particles, Whereas Neither Removal Nor Blocking of significant impact on the mDC capture of virions (Fig. 5A for B GM1 Shows Any Impact. Our data show that, whereas both α2–3- HIVLai; Fig. 5 for Gag-eGFP). linked gangliosides GM3 and GM1 are capable of mediating mDC Because we were unable to detect a change in GM1 levels on capture when overexpressed on artificial liposome particles or on virus produced from GSLhi–THP-1 cells and knockdown of GM1 virions produced from cells exogenously enriched for lipids, only had no impact on mDC capture of the virions produced, we GM3 appears to be relevant in the context of native virus. To performed blocking experiments to further verify that GM3 has further address this point, we performed additional siRNA a significant role in mDC capture of HIV-1. Virus particles were knockdowns in HEK293T cells, targeting the GM2 synthase preincubated with either cholera toxin B (CtxB) (to bind virion-

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1201104109 Puryear et al. Downloaded by guest on September 25, 2021 The selective reduction of α2–3 NeuNAc from the virion results in a marked decrease in mDC capture (Fig. 1). In contrast, when virions are exogenously enriched for α2–3 NeuNAc (Fig. 2), or artificial liposomes are created that possess this residue (Fig. 3), capture by mDCs is dramatically enhanced. Differentiation of THP-1 monocytoids to macrophages up-regulates the expression of α2–3 NeuNAc gangliosides GM3 and GM1 (Fig. 4). Impor- tantly, virions produced from these activated cells have increased levels of GM3, but not GM1, and demonstrate enhanced mDC capture and transfer (Fig. 4), which can be blocked by the addition of GM3-specific antibodies (Fig. 5). Whereas previous studies have demonstrated that HIV-1 par- ticles incorporate both α2–3 NeuNAc-linked GM1 and GM3 gangliosides (12, 18), a recent report suggests that assembly of HIV-1 Gag occurs at GM1-deficient lipid rafts (23). Our results support this recent finding and demonstrate that although GM1 can mediate mDC capture when added exogenously to virions (Fig. 2), in the absence of artificial lipid enhancement virus par- ticles derived from activated monocytes are only enriched in GM3. Interestingly, virions are clearly capable of incorporating higher levels of GM1 when GM1 alone is overexpressed (Fig. 2). Therefore, the active exclusion of GM1 from virions derived from infected THP1 monocytes may reflect the heterogeneity of GM1- and GM3-containing plasma membrane rafts and utilization of GM3-containing rafts as the preferred virus assembly site. Hence, Fig. 5. Impairment of GM3-dependent interactions of HIV-1 particle results these results demonstrate that although mDCs can recognize both in decreased capture by mDCs. (A)HIVLai or (B) Gag-eGFP VLPs produced gangliosides GM3 and GM1, it is GM3 recognition that accounts from siRNA transfected HEK293T cells were analyzed for mDC capture by (A) + for a significant portion of mDC-mediated capture of HIV-1. MICROBIOLOGY p24gag ELISA or (B) % eGFP cells by FACS. NT, nontargeting; UGCG, gluco- GM3 is known to be present in the plasma membrane of syltransferases; ST3, GM3 transferase; and B4, GM2 transferase. Data are + normalized to NT-treated particles and reported as fold decrease in mDC macrophages and activated CD4 T cells (20, 24), the primary capture. Values represent averaged data from at least two donors, ±SEM, targets of HIV-1 and therefore the primary cells from which de

*P < 0.05, one-sample t test. (C)HIVLaiΔEnv or (D) Gag-eGFP VLPs were novo virions are produced in vivo. It has been well established preincubated with increasing concentrations of α-GM3 Fab (solid gray lines) that chronic immune activation is a hallmark of HIV-1 disease. or CtxB (dashed black lines), before addition to mDCs. Isotype control Fab Furthermore, HIV-1 patients exhibit a significant overexpression was tested at the highest concentration for each assay (open circles). Virion of GM3 (25) and also develop α-GM3 antibodies (26). It is very fi gag + capture was quanti ed by (C) p24 ELISA or (D) % eGFP cells by FACS. All likely that viruses derived from activated monocytes or T cells values were normalized to the (C) virus or (D) VLP-only condition. Average in vivo would also result in enhanced incorporation of GM3 data from a minimum of two donors ±SEM are reported. within progeny virions with concurrent enhancements in DC capture and dissemination in vivo. associated GM1), or α-GM3 Fab (to bind virion-associated The viral incorporation of GM3 may aid early transmission GM3). Both conditions were compared against a mock pre- events of HIV-1 in at least two ways. First, the use of an Env- fi incubation of media only, and an isotype control Fab was tested independent binding mechanism may provide a tness advantage cis at the highest concentrations used for α-GM3 Fab. Whereas to the virus by limiting viral fusion and nonproductive -infection preincubation with increasing concentrations of CtxB had mini- of DCs. Secondly, virus binding via GM3 may help to elucidate the way in which HIV-1 is able to transit through mDCs and retain mal impact on the ability of mDCs to capture HIVLaiΔEnv C D infectivity. In this regard, HIV-1 particles share many properties particles (Fig. 5 , dotted line) or VLPs (Fig. 5 , dotted line), – preincubation with increasing amounts of α-GM3 Fab competi- with cell-derived exosomes (15, 27 29). Exosomes provide a mechanism of intercellular communication that can allow for the tively inhibited mDC capture of HIVLaiΔEnv particles (Fig. 5C, solid line) and VLPs (Fig. 5D, solid line). The control Fab exchange of membrane proteins between cells or the delivery of resulted in a modest decrease in capture of HIV ΔEnv, al- signaling proteins, mRNA and miRNA (30). Intriguingly, exom- LAI somes can also mediate cross-presentation by entering mDCs, though only α-GM3 Fab was statistically different from the mock limiting exposure to endosomal acidification (31), and ultimately condition. Of note, a higher concentration of Fab was required presenting antigen to T cells in the absence of any fusion events. to block HIVΔEnv than Gag-eGFP VLP, likely as a consequence Because exosomes directly compete with HIV-1 for mDC capture of the inherent differences in assembly and budding that exist (15) and both are enriched for GM3 (18, 32), we postulate that between Gag-GFP VLPs and full-length virus (22) that could both types of particles bind to mDCs through GM3 and that this impact the relative amounts of GM3 incorporation. interaction leads to trans-dissemination (exosomes) or trans-in- These results demonstrate that, although GM1 is physically fection (HIV). This trans-dissemination pathway, thus appears to capable of mediating mDC capture when overexpressed, it is not fi be an intrinsic mechanism of mDCs that HIV has parasitized for present in virus at suf cient levels to play a substantial role in facilitating its dissemination, while avoiding recognition as an this process. Rather, virion-associated GM3 is the principal Env- agent that should be targeted for degradation. independent ligand necessary for mDC-mediated HIV-1 capture and trans-infection. Materials and Methods Discussion Additional details are available in SI Materials and Methods. The results from this study demonstrate that mDCs can mediate Cells and Viruses. Primary cells were cultured as previously described (14). THP-1 HIV-1 capture through a particle-associated α2–3-linked sialic acid cells (ATCC; TIB-202) were cultured in RPMI, 10% (vol/vol) FBS, 1% (vol/vol) and that virion incorporation of GM3 mediates this interaction. penicillin/streptomycin. Viruses were produced from HEK293T, HeLa, or THP-1

Puryear et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 cells as described previously (14, 15, 29). Increased GM3 levels were obtained Competition Assays. VLPs or HIVLaiΔEnv were preincubated with increasing by Pam3CSK4 stimulation of THP-1 cells or exogenous enrichment of HeLa cells amounts of CtxB (Sigma; C9903) or α-GM3 DH2 Fab (derived from murine using solubilized GM3 or GM1 (Matreya). Decreased GSL levels were obtained DH2 hybridomas (33), a kind gift from Sen-itiroh Hakomori, Pacific North- through glucosyltransferase knockdowns in HEK293T cells. Sialic acid was re- west Diabetes Research Institute, Seattle, WA), or preincubated with in- moved from virions by treatment with α2–3, α2–6, α2–8 neuraminidase (NEB; creasing amounts of liposomes before performing mDC virus capture assays. P0230S) or α2–3 neuraminidase (NEB; P0728S). ACKNOWLEDGMENTS. We thank Drs. Greg Viglianti and Andy Henderson, and members of the Gummuluru laboratory for discussion and critical review Liposomes. Liposomes 130 nM in diameter comprised 54% DPPC, 1% PS, and of the manuscript; Dr. Sen-Itiroh Hakamori for the gracious gift of the α-GM3 fi + 45% cholesterol. Modi cations included 1% Cer, GalCer, GM3, GM1, or GQ1b hybridoma; Dr. Andrea Cimarelli for the generous gift of plasmid pSIV3 ; and (Table S1) and fluorescently tagged with TopFluor cholesterol (Avanti Polar the Boston University Medical Campus flow cytometry core facility for tech- Lipids; 810255). nical assistance. We thank the NIH AIDS Research and Reference Reagent Program for providing us with the following reagents: recombinant human DC Capture and Transfer Assays. Virus (40 ng p24gag unless noted otherwise), IL-2 (contributed by Roche), HIV-1 immunoglobulin (cat. no. 3957; contrib- uted by Dr. Luis Barbosa), and α-p24gag hybridoma (clone 183-H12-5C; con- VLPs (2 ng p24gag), or liposomes (5 μL) were incubated for 1 h at 37 °C with + tributed by Drs. Kathy Wehrly and Bruce Chesebro). This study was supported mDCs and assayed for capture or transfer to autologous CD4 T cells as by National Institutes of Health (NIH) Grants AI064099 (to S.G.) and AI081596 previously described (14). (to S.G.), and National Research Service Award F32 AI084558 (to W.B.P.).

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