Molecular Events Underlying Cell-Associated HIV Transmission. Rahm Gummuluru Boston University School of Medicine Department of Microbiology

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1 Molecular Events Underlying Cell-Associated HIV Transmission Rahm Gummuluru Boston University School of Medicine Department of Microbiology

conjugates (Macdonald et al, 23) DC T cell Immunological Synapse DC T cell Virological Synapse HIV DC T cell MHC TCR (CD3) CD4 or CD8 ICAM1

2 HIV Dendritic Cell Interactions Robust virus replication in dendritic cell (DC) T cell co-cultures (Cameron et al, 1992). Mature DCs can transfer HIV-1 particles to CD4 + T cells at an enhanced efficiency primarily because of establishment of stable DC T cell conjugates (Macdonald et al, 23) DC T cell Immunological Synapse DC T cell Virological Synapse HIV DC T cell MHC TCR (CD3) CD4 or CD8 ICAM1 LFA1 CD8/86 CD28 EJI 25 35:1741 MHC TCR (CD3) CD4 or CD8 ICAM1 LFA1 CD8/86 CD28 CD81 Putative evasion from neutralizing antibody responses (Sagar et al, 212)

3 Dendritic Cells Facilitate HIV-1 Infection! DC T cell co-cultures are sites of robust viral replication# Kinetics of Establishment of Productive Infection in CD4 + T cells is Enhanced in the Setting of DC T cell Co-cultures# Magnitude of Virus Replication in # CD4 + T cells is Enhanced in the Setting of DC T cell Co-cultures # Time (min) T cells alone# DC T cell # T cells alone# DC T cell# DCs alone# DC - T transfer (5% fusion) = 38.6 ± 1.75 min T cells alone (5% fusion) = 84 ± 2.5 min J. Virol : "

Dendritic Cells: Hostile Cellular Environment for HIV

endocytosis Intrinsic Responses CD4 CCR5 Produc've

receptors X Fusion & Entry Nuclear Import X Mx2

Apobec3G Virus Par'cle Degrada'on Virus Sensing An'gen

4 Dendritic Cells: Hostile Cellular Environment for HIV Innate Responses Phagocytosis Receptor mediated endocytosis Intrinsic Responses CD4 CCR5 Produc've Infec'on Scavenger receptors Lysosome PRRs C type lec'n receptors X Fusion & Entry Nuclear Import X Mx2 Integra1on Uncoa1ng X Reverse Transcrip1on SamHD1 Apobec3G Virus Par'cle Degrada'on Virus Sensing An'gen presenta'on Cytoplasmic Sensor of Gag (?) Viral protein synthesis X Transcrip1on Assembly & Release

Captured HIV-1 Particles transferred to T

5 Dendritic Cell-Associated HIV-1 trans Infection CD4 + T cells CD4 + T cells HIV-attachment factors Concentration within Membrane Invaginations MVB Captured HIV-1 Particles transferred to T cells Without de novo virus replication in Dendritic Cells

6 Dendritic Cell-Associated HIV-1 trans Infection CD4 + T cells CD4 + T cells HIV-attachment factors Concentration within Membrane Invaginations MVB Captured HIV-1 Particles transferred to T cells Without de novo virus replication in Dendritic Cells

7 HIV-1 Attachment factors on Dendritic Cells Envelope Glycoprotein gp12 - Dependent Binding C-type lectin receptors - Recognition of the glycosylated gp12 - DC-SIGN, Langerin, DCIR Heparan sulfate proteoglycans (Syndecans) - Polyanionic surface - Charge-based interactions with gp12 Geijtenbeek et al, Cell 2; Turville et al, Nat. Immunol 22; de Witte et al, PNAS 27; Lambert et al, Blood 28;

8 Envelope Glycoprotein-Independent Capture of HIV-1 Particles by Dendritic Cells HEK293T cell derived virus Jurkat T cell derived virus HIV 1 (wt) HIV 1/Δenv Immature DCs Mature DCs Immature DCs Mature DCs e r tu p a C gag p2 4 % Macrophage drived virus 4 CD4+ T cell derived virus HIV 1 (wt) HIV 1/Δenv Immature DCs Mature DCs Immature DCs Mature DCs Env deficient virus par'cles derived from divergent cellular sources remain competent for capture by dendri'c cells (J. Virol : )

9 ~ 7 14 Env trimers per par1cle Zhu et al., Nature 26 Sougrat et al., PLoS Pathogens 27 Host cellular determinants incorporated in virus particle membrane might play a role in HIV capture by dendritic cells.

HIV Incorporates Molecules from Host Membrane Lipid Rafts Binding galactoyslceramide sphingomyelin cholesterol proteins glycosphingolipid Fusion Lipid Rafts

cfm Modified from Higashi, Glycoworld Lipid and protein composition of the virus particle membrane is determined by the site of virus assembly and exit.

10 HIV Incorporates Molecules from Host Membrane Lipid Rafts Binding galactoyslceramide sphingomyelin cholesterol proteins glycosphingolipid Fusion Lipid Rafts Budding Modified from progenics.com/hiv.cfm Modified from Higashi, Glycoworld Lipid and protein composition of the virus particle membrane is determined by the site of virus assembly and exit. Virus particles enriched in cholesterol, sphingolipids, glycosphingolipids, and lipid raft associated cellular proteins such as CD59 and tetraspanin proteins. Strategy to identify HIV determinant necessary for capture by DCs Selective depletion of virus particle membrane constituents

HIV Proteome Tetraspanins GPI-linked proteins Class II MHC HIV Lipidome Cholesterol Sphingomyelin Glycosphingolipids Removal of

11 Virus hat Virus Particle is the Particle-Associated Glycosphingolipids W aptu HIV par'cle determinant Proteins necessary are for DC not a c Required Essential for for DC HIV-1 Mediated Capture HIV-1 by Capture DCs re re? HIV Proteome Tetraspanins GPI-linked proteins Class II MHC HIV Lipidome Cholesterol Sphingomyelin Glycosphingolipids Removal of Protein from the virion Removal of GSLs from the virion mock Pronase treated mock GSL deficient Izquierdo-Useros, Blood 29 Hatch, J.Virol 29

Maturation of DCs Enhances Env-Independent Glycosphingolipid Mechanism of HIV-1

Pathway PDMP Ceramide X Glucosylceramide Glycosphingolipids Gag egfp VLP Capture 1

1 1 2 1 3 1 4 FL1-H HIV VLPs produced in the presence of PDMP are impaired for capture

12 Maturation of DCs Enhances Env-Independent Glycosphingolipid Mechanism of HIV-1 Capture HIV wt Gag egfp myr p2 p1 MA CA NC p6 egfp dihydroceramide GSL Biosynthesis Pathway PDMP Ceramide X Glucosylceramide Glycosphingolipids Gag egfp VLP Capture 1 Immature DCs Mature DCs 75 Galactosylceramide Sphingomyelin 5 Gag egfp 25 CTxB IP FL1-H HIV VLPs produced in the presence of PDMP are impaired for capture by mature DCs Hatch et al, J. Virol : ; Izquierdo Useros et al, Blood :

13 Glycosphingolipids (GSLs): Raft-Resident Lipids Lipid por'on of GSL, ceramide, is embedded in the outer leaflet of the plasma membrane, and their glycan moie'es project into the extracellular space. GSL Biosynthesis Pathway

14 GM3-Depleted Virions are Deficient for Capture by Mature DCs GSL Biosynthesis Pathway dihydroceramide UGCG Ceramide Glucosylceramide Lactosylceramide Galactosylceramide Sphingomyelin ST3GalV GM3 GM3 NT UGCG ST3GalV *** ***.72 (+/-.15).37 (+/-.7) *** GM3/p24 (+/-SD).33 (+/-.15 * ) ** Puryear et al, PNAS :

15 GSL Biosynthesis Pathway dihydroceramide SPHINGOLIPIDS SPHINGOMYELIN (major mb phospholipid) Sphingomyelin synthase ER-golgi transport CERT FB1 Galactosyl transferase / GalCer synthase CERA MIDE Glucosyl transferase / GCS PDMP SULFATIDES,GM4 GLYCOSPHINGOLIPIDS Lactosyl Ceramide Blood group: lactoseries and neolactoseries FAPP2 ER-golgi transpor t Glucosyl Ceramide Lactosyl transferase GM3 a-series ST3GAL5 GD3 b-series X GT3 c-series Gangliosides (sialic-acid-containing GSLs ) B4GALT asialo-gsls globosides A4GALT B3GNT5 Lac3Cer GA2 Gb3 B4GALT B4GALT B4GALT GM2 GD2 GT2 GA1 GM2α Gb4 GalGb3Cer GM1 GD1 GT1 others others others others others others others

16 Liposome system for analyzing HIV Capture by Mature DCs 15nm (~ virus sized) par1cles cholesterol coupled to TopFluor dye HIV Mock (PS) +Lipid Composi1on ~ 45% cholesterol >3% phospholipids ~1% PS.5 3.5% GM3 Composi1on 45% cholesterol* 54% DPPC 1% PS Composi1on 45% cholesterol* 53% DPPC 1% PS 1% Lipid

17 Inclusion of GM3 in Virus-sized Liposomes Results in Enhanced Mature DC capture Mock (PS) 3 p =.2 *** 2 1 +Lipid PS Cer Gal GM3 GQ1b Type of liposome NeuAc Gal NeuAc NeuAc Gal Glc NeuAc Gal GalNAc Gal Glc ceramide GalCer GM3 (α2,3-sialic acid) GQ1b (α2,8-sialic acid)

18 Blocking GM3 Results in Attenuated Capture of HIV by Mature Dendritic Cells GFP Competition With Liposomes GFP Competition With α-gm3 Fab fragments

19 Immune Activation and GM3 Enrichment in HIV-1 Particles GM3 is the major ganglioside in the plasma membrane of lymphocytes and monocytes GM3 synthase activity is upregulated upon immune activation, and upon initiation of monocyte to macrophage differentiation

GM3 enrichment in Virus Producer Cells Produces HIV that is Captured by DCs

Macs Monocyte/Pam 3 CysK 4 mock untreated enriched mock untreated enriched

GM3(Alexa488) 1 1 1 2 1 3 1 FL1-H GM3(Alexa488) 1 4 2.

20 GM3 enrichment in Virus Producer Cells Produces HIV that is Captured by DCs with Enhanced Efficiency GM3 Expression on Virus Producer Cells HeLa Monocyte Macs Monocyte/Pam 3 CysK 4 mock untreated enriched mock untreated enriched mock untreated enriched FL1-H GM3(Alexa488) GM3(Alexa488) FL1-H GM3(Alexa488) HIV-1 Capture by Dendritic Cells mock +GM3 Enrichment NTC +Pam3CysK4 THP Treatment (HIV Lai )

21 Summary: GM3 mediates HIV capture by DCs Loss of Function - Knockdown of GM3 synthase results in decreased capture HIV Gain of Function -Enrichment with GM3 (immune activation or lipid addition) results in enhanced capture -Liposomes with GM3 have enhanced capture HIV Liposome Competition Assays - GM3 liposomes compete for capture - α-gm3 Fab blocks capture Puryear et al, PNAS :

22 What is the DC receptor responsible for GM3 dependent HIV 1 capture?

23 Characteristics of the GM3-Dependent HIV Capture Mechanism on Dendritic Cells 1. Pronase Sensi1ve IFN α Inducible None (immature DCs) Pam 3 CysK4 Poly(I:C) LPS IFN! TNF! Mock Pronase DC pretreatment Dendritic Cell Stimulation 3. Myeloid Cell Specific 4. α2,3 Sialic Acid Requirement 5 4 NT + IFNα 3 2 ** ** 1 Neuraminidase Treatment of HIV

24 SIGLEC Family of Lectins Sialic acid-recognizing Ig-superfamily lectins Major homologous family of I-type lectins divided into 2 sub-groups Evolutionary conserved sub-group (Siglecs-1, -2 and -4) Rapidly evolving CD33 and Siglec-3-related subgroup (Siglecs-3 and in primates) Broadly expressed in cells of the immune system

25 SIALIC-ACID-BINDING IMMUNOGLOBULIN-LIKE LECTINs cdcs Siglec1 (CD169) expressed exclusively on myeloid cells expression induced by type I IFN binds to terminal α2,3-linked sialic acid residues

26 Maturation of Dendritic Cells Enhances CD169 Expression Monocyte Day 2 Day 4 Immature DCs (day 6) Mature DCs (LPS) CD169 Quantitative FACS HIV-1 Capture p=

27 Selective Depletion of CD169 in Mature Dendritic Cells Abrogates HIV-1 Capture Lentivector/shRNA-mediated knockdown of CD169 or DC-SIGN in mature DCs No Stain Isotype Control Scrambled shrna CD169 shrna DC-SIGN shrna DC-SIGN CD169 CD86

28 Neutralizing Antibodies Against CD169 block DC Mediated HIV-1 Capture & Trans Infection Virus Binding HIV-1 trans Infection CD169 DC-SIGN CD169/DC-SIGN Isotype (IgG1) Isotype (IgG2b) Isotype (both) Non-treated Immature Mature Immature Mature

Exogenous Expression of CD169 in Receptor-Naïve Cells Rescues GSL-Dependent HIV-1 Capture & Trans Infection Express human CD169 in Raji B cell line

Raji/CD169 8 6 4 2 1 1 1 CD16 9 1 2 FL1- H 1 3 1 4 CD169 actin 8 6 HIV-1 Capture HIV/Lai HIV/LaiΔenv 6 5 HIV-1 Capture 3 25 HIV-1 Trans Infection 4 4 3 NT

29 Exogenous Expression of CD169 in Receptor-Naïve Cells Rescues GSL-Dependent HIV-1 Capture & Trans Infection Express human CD169 in Raji B cell line (non-permissive for HIV capture) Incubate with WT virus (NT), virus deficient in GSL (PDMP), or Env-deficient virus Co-culture with T cells 1 Raji Isotype Raji/CD CD FL1- H CD169 actin 8 6 HIV-1 Capture HIV/Lai HIV/LaiΔenv 6 5 HIV-1 Capture 3 25 HIV-1 Trans Infection NT PDMP 2 15 NT PDMP Raji Raji/CD169 Raji Raji/CD169 Raji Raji/CD169 Capture and transfer of GSL-deficient HIV-1 particles by Raji/CD169 cells is attenuated

30 GM3 Recognition by CD169 is Essential for HIV-1 Capture 25 Liposome Capture Assay 1.2 Liposome Compe11on Assay 2 15 p=.3 p=.18 Raji Raji/CD Blank GM GalCer GM3 GM1 GQ1b Volume (µl) 1

abrogates sialic acid binding Vinson M et al, JBC 1996 A CD169 3 2 C HIV 1 Capture ** **

31 Lack of HIV-1 Capture by Cells Expressing CD169 Mutant Deficient for Sialic Acid Recognition WT = human CD169 R96A = no affect on sialic acid binding R116A = abrogates sialic acid binding Vinson M et al, JBC 1996 A CD C HIV 1 Capture ** ** Mock CD169 R96A R116A actin B Cell Surface CD169 Expression 1 Unstained Isotype WT R96A R116A 3 D *** 2 1 CD169

32 Murine CD169 can Mediate Retroviral Capture and Trans Infection Muri ne Murine CD169 expressio Expression n als o HIV-1 seen Capture on m yeloid cell smlv Capture 1 Unstained 5 Isotype 72% ntity between hum an nd m 4 M o o u ck se Murine CD169 Murine CD169 id 8 e- bo 6t h type I transm aemb3 ran prote CD1698 Mu i r n ine s CD169 6 e 2 4 he H similarity in th llu 4 e ex 1 t race lar domain i 2 17 Ig like g d 1 2 om 1 3 a 4 in 1 s 5 (N 1 -terminal N V T -set d G o S m L-/- ain and 16 C N - T set dos G m SL a -/- ins) CD169 HIV-1 Transfer MLV 2 Sialic acid binding site is ical between human a nd mouse Transfer CD169 1 t i 1 d e nt 8 8 GM3 gangliosides are enr 6 i c h ed in d bilayer of 6 s i mple retr ovirus, MLV lipi Chan et al, J. Viro l Mock 2 2 Mock Murine CD169 Mock Murine CD169

33 HIV Gag-mCherry VLPs Co-localize with CD169 on Mature DC Surface and at the DC T cell Virological Synapse No VLP VLP < 1min VLP 12minDC : T cell Correlation Coefficient.2 (+/-.2).45 (+/-.18).7 (+/-.17).84 (+/-.1)

Localization of HIV-1 Particles in CD169 +

Super-resolution Microscopy HIV-1 Particles

34 Localization of HIV-1 Particles in CD169 + Plasma Membrane Invaginations of Myeloid DCs MDC# HIV CD169 X Y X CD169# HIV# MDC# Z X CD169# HIV# Deconvolution Microscopy 5 nm Z X Super-resolution Microscopy HIV-1 Particles Captured by CD169 are Trafficked to Non-Lysosomal Compartments#

35 HIV-1 Evasion of Adaptive Immune Responses HIV VRCO1 Surface HIV VRCO1 Permeabilized.2. All Surface VRC1 HIV-1 CD169 HIV-1 CD Surface Permeabilized. All Surface CD169

36 Conclusions Expression of CD169 on monocyte-derived DCs, inflammatory DCs and blood myeloid DCs is required for GM3-dependent HIV-1 capture. Blocking CD169 with neutralizing antibody or depletion via shrna results in loss of DC-dependent HIV-1 capture and trans infection. Induced expression of CD169 in a non-permissive cell confers the ability to capture and transfer virus. Mutation that eliminates sialic acid binding (R116A) results in attenuation of virus capture by CD169. HIV-bound CD169 re-localizes to a polarized region of DC and is colocalized with HIV-1 particles at the DC T cell infectious synapse. HIV-1 particles captured by CD169 are trafficked to plasma membrane invaginations Invariably accessed by surface-applied anti-gp12 monoclonal antibodies Puryear & Akiyama et al, PLoS Path 213 Izquierdo-Useros et al, PLoS Biology 212

37 CD169 + DC Mediated HIV-1 Trans Infection! Dependent on a host cell- derived ligand (GM3) and receptor (CD169) Parasi1za1on of cell- to- cell recogni1on mechanism by HIV for virus dissemina1on Protec1on from innate and adap1ve immune responses de novo virus Env dependent DC- SIGN CD4 & CoR infecpon blocked Virus producer cell Immune Ac1va1on GM3 Incorpora'on in Virus Par1cles GM3 dependent Syndecan GSLs CD169?? endocytosis Dendri9c cell degradapon Trans infecpon T cell Immune Ac1va1on DC- mediated HIV trans infec1on

Past Members Steve Hatch Hiren Patel Erica Li University of Pittsburgh Zandrea Ambrose Boston

38 Acknowledgements Boston University Chemistry & Photonics Björn Reinhard Xinwei Yu Amin Feizpour Vutara, Inc Manasa Gudheti Wendy Puryear Hisashi Akiyama Nora Ramirez Suzanne Geer Caitlin Miller Past Members Steve Hatch Hiren Patel Erica Li University of Pittsburgh Zandrea Ambrose Boston University School of Medicine Manish Sagar Deborah Anderson Tsuneya Ikezu Funding: NIH RO1-AI6499, R56-AI14393

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