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									                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              *Department of Microbiology
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Boston University


                            271                                                  Immature Dendritic Cell-Derived Exosomes can Mediate HIV-1 Trans Infection                                                                                                                                                                                                                                                                                                                                                                                                                        School of Medicine
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                R-509, 715 Albany Street,
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Boston, MA 02118
                                                                               Rebecca D.Wiley*, and Rahm Gummuluru*; Boston University School of Medicine, Boston, MA                                                                                                                                                                                                                                                                                                                                                                                                           email: rdwiley@bu.edu,
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   rgummulu@bu.edu


                                                                                                                                                                            Figure 3. Kinetic analysis of endocytosed                                                                                                                                  Exosomes                                                                                                                                             Figure 7. Infectivity potential of
Background: Immature dendritic cells (DCs) can capture human
                                                                                                                                                                                          virus particles                                                                          • Present in multivesicular endosomal bodies (MVB).                                                                                                                                                                         exosome-associated HIV
immunodeficiency virus type 1 (HIV-1) and mediate transfer of virus                                                                                          DCs were incubated with virus particles (10ng p24gag) for 2 h at 37oC, washed, treated                                • Formed by invagination and budding from the limiting
                                                                                                                                                              with 0.25% trypsin, and plated. Cell supernatants and cell lysates were collected at                                                                                                                                                                                                                    Whole supernatant, HLA-DR1+, or CD14+                                              Cell-free virus (Lai-luc) or HLA-DR1+
                                                                                                                                                                        various time points and analyzed for p24gag content by ELISA
                                                                                                                                                                                                                                                                                     membrane of MVBs, and contain cytosolic components.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                   fractions from 24h supernatants from Lai-luc                                       fractions from 24h supernatants of Lai-luc
particles to T cells. The mechanism by which this transfer occurs                                                                                                                                                                                                                  • Released upon fusion of MVB with the plasma membrane.                                                                                                                           exposed DCs were incubated with MAGI-                                             exposed DCs were incubated with MAGI-
                                                                                                                                                                                                   Lai (CXCR4-tropic)                   Ba-L (CCR5-tropic)                         • Secreted by DCs.                                                                                                                                                                       CCR5 cells. Blue foci were                                                        CCR5 cells. Blue foci were
however remains unclear.                                                                                                                                               12                                                          10                                                                                                                                                                                                                                           counted 2 days later.                                                             counted 2 days later.
                                                                                                                                                                                                                                                                                   • Have densities around 1.13 g/ml.




                                                                                                                                           % virus retained (p24gag)
                                                                                                                                                                                                                                                                                   • Have been shown to express antigen presenting molecules                                                                                                                                                120                                                                        1000
                                                                                                                                                                       9                                                            8




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Fold enhancement of
                                                                                                                                                                                                                                                                                     (MHC I, MHC II;HLA-DR1, and CD1),tetraspanins (CD9,CD63,CD81),




                                                                                                                                                                                                                                                                                                                                                                                                                                                                     Relative infectivity
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            100




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   virus infectivity
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        100
Methods: DCs were derived from CD14+ monocytes by                                                                                                                                                                                                                                    adhesion molecules (CD11b and CD54), and costimulatory                                                                                                                                                   80
                                                                                                                                                                        6                                                           5                                                molecules (CD86).
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              60                                                                         10
differentiation in GM-CSF and IL-4 containing media. Kinetic
                                                                                                                                                                        3                                                           3                                                                                                                                                                                                                                                         40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1
experiments were performed using CXCR4- (Lai) or CCR5- (NL4-                                                                                                                                                                                                                                                                                                                                                                                                                                  20
                                                                                                                                                                        0                                                           0                                                                     Figure 6. HIV-1 particles released from DCs are                                                                                                                                      0                                                                        0.1
                                                                                                                                                                                                   0        2      10        24         0         2        10    25
3/Ba-L) tropic virus isolates. Luciferase expressing HIV-reporter                                                                                                                                                                                                                                                    associated with exosomes                                                                                                                                                          le
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         +            +




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  t io 1 +
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 ru ee
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    ho ’t               R            14




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               ac R
                                                                                                                                                                                                             Time (h)




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               vi l-fr
                                                                                                                                                                                                                                                      Time (h)                                                                                                                                                                                                                                                     -D            D




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             fr A-D

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   W up                      C




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    s
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 LA




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  el
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     s




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                C




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 L
                                                                                                                                                                                                       Cell-associated p24gag               Cell-associated p24gag                                                                                                                                                                                                                                           H




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               H
viruses were used in single cycle of replication assays. Exosomes                                                                                                                                                                                                                                         24 h supernatant from Lai exposed DCs was subject to differential sucrose gradient
                                                                                                                                                                                                                                                                                                          centrifugation. Gradient fractions were collected and analyzed by Western blot for
                                                                                                                                                                                                       Cell-free p24gag                     Cell-free p24gag                                                            HLA-DR1 expression, and for p24gag content by ELISA.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                   All infectivity associated with the 24h supernatants
from virus-exposed DC supernatants were isolated via antibody-
                                                                                                                                                                                                                                                                                                                                                                                                                                                                       from Lai-luc exposed DCs could be immuno-




                                                                                                                                                                                                                                                                                                                                                          Lai/DC
                                                                                                                                          HIV-1 particles (p24gag) captured by DCs can escape                                                                                    MW                                     fraction number                                                                                               fraction number
conjugated magnetic beads or by differential centrifugation and




                                                                                                                                                                                                                                                                                                         Lai
                                                                                                                                          degradation (~30%) and are subsequently released
                                                                                                                                                                                                                                                                                (kDa)                          7 8     9 10 11 12 13 14 15 16 17 18
                                                                                                                                                                                                                                                                                                                                                                                                                       0.7
                                                                                                                                                                                                                                                                                                                                                                                                                             7 8 9 10 11 12 13 14 15 16 17 18         isolated by HLA-DR1+ magnetic beads. These
                                                                                                                                                                                                                                                                                88
floatation on linear sucrose gradients. Exosome preparations were                                                                                      into the extracellular mileu                                                                                                                                                                           HLA-DR1
                                                                                                                                                                                                                                                                                                                                                                α/β
                                                                                                                                                                                                                                                                                                                                                                                                                       0.6                                            fractions are 50-fold more infectious per ng of




                                                                                                                                                                                                                                                                                                                                                                                                      p24gag CA (ng)
                                                                                                                                                                                                                                                                                                                                                                                                                       0.5
                                                                                                                                                                                                                                                                                50
                                                                                                                                                                                                                                                                                                                                                                                                                       0.4                                                   p24gag than cell-free virus stocks.
analyzed for exosomal proteins by western blotting, examined for                                                                                                                                                                                                                                                                                                                                                       0.3
                                                                                                                                                                                                                                                                                33                                                                            HLA-DR1
                                                                                                                                                                        Figure 4. Nature of p24gag released into the                                                            27                                                                               α
                                                                                                                                                                                                                                                                                                                                                                                                                       0.2
p24gag content by ELISA, or used in infectivity transfer studies with                                                                                                                                                                                                                                                                                                                                                  0.1
                                                                                                                                                                               extracellular mileu over time                                                                                                                                                                                                                                                                                        Proposed model of exosome mediated




                                                                                                                                                                                                                                                                                                   1.2 42
                                                                                                                                                                                                                                                                                                   1.1 65
                                                                                                                                                                                                                                                                                                   1.0 70


                                                                                                                                                                                                                                                                                                   1.1 25
                                                                                                                                                                                                                                                                                                   1.1 10
                                                                                                                                                                                                                                                                                                   1.1 22


                                                                                                                                                                                                                                                                                                   1.2 19
                                                                                                                                                                                                                                                                                                   1.2 5


                                                                                                                                                                                                                                                                                                   1.2 6
                                                                                                                                                                                                                                                                                                   1.1 26




                                                                                                                                                                                                                                                                                                   1.2 77



                                                                                                                                                                                                                                                                                                         7




                                                                                                                                                                                                                                                                                                                                                                                                                       1.2 42
                                                                                                                                                                                                                                                                                                                                                                                                                       1.1 22



                                                                                                                                                                                                                                                                                                                                                                                                                       1.2 05


                                                                                                                                                                                                                                                                                                                                                                                                                       1.2 6
                                                                                                                                                                                                                                                                                                                                                                                                                       1.0 70
                                                                                                                                                                                                                                                                                                                                                                                                                       1.1 26
                                                                                                                                                                                                                                                                                                                                                                                                                       1.1 5
                                                                                                                                                                                                                                                                                                                                                                                                                       1.1 10


                                                                                                                                                                                                                                                                                                                                                                                                                       1.1 5
                                                                                                                                                                                                                                                                                                                                                                                                                       1.2 19


                                                                                                                                                                                                                                                                                                                                                                                                                       1.2 77



                                                                                                                                                                                                                                                                                                                                                                                                                             7
                                                                                                                                                                                                                                                                                                       00


                                                                                                                                                                                                                                                                                                       33


                                                                                                                                                                                                                                                                                                      44




                                                                                                                                                                                                                                                                                                                                                                                                                           33
                                                                                                                                                                                                                                                                                                                                                                                                                           02



                                                                                                                                                                                                                                                                                                                                                                                                                           56
T cells or MAGI/CCR5 cells.




                                                                                                                                                                                                                                                                                                       4




                                                                                                                                                                                                                                                                                                                                                                                                                          44
                                                                                                                                                                                                                                                                                                       5
                                                                                                                                                                                                                                                                                                       6


                                                                                                                                                                                                                                                                                                       0
                                                                                                                                                                                                                                                                                                       2
                                                                                                                                                                                                                                                                                                       4


                                                                                                                                                                                                                                                                                                       8
                                                                                                                                                                                                                                                                                                      8




                                                                                                                                                                                                                                                                                                      1




                                                                                                                                                                                                                                                                                                                                                                                                                           4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          trans infection of T cells




                                                                                                                                                                                                                                                                                                                                                                                                                           4



                                                                                                                                                                                                                                                                                                                                                                                                                           0
                                                                                                                                                                                                                                                                                                                                                                                                                           6
                                                                                                                                                                                                                                                                                                                                                                                                                           8


                                                                                                                                                                                                                                                                                                                                                                                                                           2



                                                                                                                                                                                                                                                                                                                                                                                                                           8


                                                                                                                                                                                                                                                                                                                                                                                                                          1
                                                                                                                                                                                                                                                                                                   1.0




                                                                                                                                                                                                                                                                                                                                                                                                                       1.0
                                                                                                                                                                                                            Lai-luc                                                                                                         Density (g/mL)                                                                                            Density (g/mL)
                                                                                                                                                                                                         (CXCR4-tropic)           SFV-luc
                                                                                                                                                                                                   106                                                                                                                                                                                                                                                                                      HLA-DR1
                                                                                                                                                                                                                                                                                                        HIV-1 p24gag expression overlaps with the density                                                                                                                                   CD9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 8. Infection of T cells by HIV-1
                                                                                                                                                                                                                                                                                                        profile for DC-derived exosomes represented by                                                                                                                                      CD1b
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          positive exosomes
                                                                                                                                                                            relative light units




                                                                                                                                                                                                   105                                                                                                                                                                                                                                                                                                       Immature
       Figure 1. DCs phenotype following HIV-1 infection                                                                                                                                                                                                DC                                              HLA-DR1 expression (1.12-1.15 g/ml) suggesting                                                                                                                                                          DC
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      7. Release of HIV-1 associated
                                                                                FACS Analysis                                                                                                      104
                                                                                                                                                                                                                                                        DC-T cell                                       vesicle (exosome)-association of HIV-1 particles                                                                                                                                                                                        internal vesicles
                                                                                                                                                                                                                                                        co-cultures                                                                                                                                                                                                                                                                             (exosomes) into
                                                                                                                                                                                                                                                        DC supernatants -                                              exocytosed by DCs                                                                                                                                                                                                   extracellular environment
                                                    CD11c            DC209     HLA-DR1          CD83            CD86                                                                                                                                    T cell cultures
                                                                                                                                                                                                   103                                                                                     DC supernatants harvested 24 h post- DC                                                     DC supernatants harvested 24h post-cell                                      1.                      HIV-1 binding
                                                          39.7          45.1         9.6           7.4            21.8                                                                                                                                                                        exposure to Lai-luc or SFV-luc were                                                    exposure to Lai env pseudotyped virus were
                                                                                                                          Immature DC
                                                                                                                                                                                                                                                                                             incubated with HLA-DR1 conjugated                                                      incubated with magnetic beads conjugated to
Relative cell number




                                                                                                                            infected                                                               102                                                                                     magnetic beads. HLA-DR1+ fractions were                                                    various antibodies. Positive fractions were
                                                                                                                                                                                                                                                                                           incubated with Jurkat T cells. Cells were                                                  incubated with MAGI-CCR5 (contain β-Gal
                                                                                                                                               DCs were exposed to Lai-luc or Semliki Forest Virus SFV-luc (MOI = 0.001), washed, and                                                         lysed 3 days later and analyzed for                                                       reporter gene controlled by HIV-1 LTR).
                                                          35.2         51.9          8.5           5.1            11.0                         placed in culture by themselves or in co-culture with Jurkat T cells. Supernatants, from                                                                                                                                                                                                                                                        2. endocytosis
                                                                                                                          Immature DC                                                                                                                                                                 luciferase activity.                                                             Infected cells were counted 2 days later.
                                                                                                                           uninfected            infected DCs cultured alone, were harvested 24 h post-virus exposure, clarified by
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    6. Fusion of MVB
                                                                                                                                                 centrifugation, and incubated with Jurkat T cells. Cells were harvested 3 days later,
                                                                                                                                                                                                                                                                                                                                                                                                                                                 DC supernatants                                                                 with plasma membrane
                                                                                                                                                                       lysed, and assayed for luciferase activity.                                                                                                                                                                                                                                  from cells
                                                                        58.5         30.6          24.1            67.3                                                                                                                                                                                                       DC supernatants                                                                                                     trypsinized 3h
                                                           87.8                                                                                                                                                                                                                                                                                                                                                          DC supernatants
                                                                                                                          Immature DC                                                                                                                                                                                         IP: anti-HLA-DR1                                                                                                    post infection
                                                                                                                          LPS activated                                                            HIV-1 particles released into cell-free                                                              25000                                                                            1000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   5. MVB containing vesicles
                                                                                                                                                                                                                                                                                 relative light units
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        with exposed HIV-1
                                                                                                                                                                                                       supernatants are infectious




                                                                                                                                                                                                                                                                                                                                                                        Infectious units/ml
                                                                                                                                                                                                                                                                                                        20000
                                                                          Mean Fluorescence Intensity                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    virions at their cell
                                                                                                                                                                                                                                                                                                                                                                                              100                                                                                                                                                                                              surface
                                                                                                                                                                                                                                                                                                        15000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       3. Internalization
                                                          DCs maintain an immature phenotype                                                                                                                                                                                                            10000                                                                                                                                                                           into endosome
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               4. Invagination & budding of
                                                                                                                                                                        Figure 5. Electron Microscopy analysis for                                                                                                                                                                             10                                                                                                                                                                             endosomal limiting membrane
                                                               24 h post-virus exposure
                                                                                                                                                                                      HIV-1 particles                                                                                                    5000

                                                                                                                                                                                                                                                                                                               0                                                                                  1
                                                                                                                                                                                                                    A




                                                                                                                                                                                                                                                                                                                                                                                                                              1b
                                                                                                                                                                                                                                                                                                                                                                                                                               14
                                                                                                                                                                                                                                                                                                                        k




                                                                                                                                                                                                                                                                                                                                                                                                                                1
                                                                                                                                                                                                                                                                                                                                                                                                       Ig




                                                                                                                                                                                                                                                                                                                                                                                                                                                             R1
                                                                                                                                                                                                                                                                                                                                                                                                                                            9


                                                                                                                                                                                                                                                                                                                                                                                                                                                    14
                                                                                                                                                                                                                                                                                                                     oc                              uc




                                                                                                                                                                                                                                                                                                                                                                                                                             DR
                                                                                                                                                                                                                                                                                                                                     uc




                                                                                                                                                                                                                                                                                                                                                                                                                                          CD
                                                                                                                                                                                                                                                                                                                                                                   IP                                                                                                                                                        Conclusions




                                                                                                                                                                                                                                                                                                                                                                                                                            CD
                                                                                                                                                                                                                                                                                                                                                   -l




                                                                                                                                                                                                                                                                                                                                                                                                                            CD
                                                                                                                                                                                                                                                                                                                                                                                                   e
                                                                                                                                                                                                                                                                                                                                  i-l




                                                                                                                                                                                                                                                                                                                                                                                                                                                  CD


                                                                                                                                                                                                                                                                                                                                                                                                                                                              D
                                                                                                                                                                                                                    Plasma                                                                                          m




                                                                                                                                                                                                                                                                                                                                                                                                us



                                                                                                                                                                                                                                                                                                                                                                                                                           A-




                                                                                                                                                                                                                                                                                                                                                                                                                                                           A-
                 Figure 2. Fate of HIV-1 particles captured by DCs                                                                                                                                                                                                                                                              La             V
                                                                                                                                                                                                                                                                                                                                             SF




                                                                                                                                                                                                                                                                                                                                                                                              Mo




                                                                                                                                                                                                                                                                                                                                                                                                                        HL




                                                                                                                                                                                                                                                                                                                                                                                                                                                         HL
                                                                                                                                                                                                                    Membrane
                            following trypsin treatment                                                                                                                                                                                                                     B
                                                                                                                                          DCs were exposed to Lai
                                                                                                                                                                                                                                                                                                                   Infectious HIV-1 Lai virus particles from DC
                                                                                                                                                                                                                                                                                                                                                                                                                                                                   1. HIV-1 particles captured by immature DCs can be
                                                                                                                                          for 3 h and fixed for thin
                                                                                                                                          section electron
                                                                                                                                                                                                                                                                                                                            supernatants are directly                                                                                                                 transmitted to T cells, without de novo infection, by
                                                          Lai (CXCR4-tropic)           Ba-L (CCR5-tropic)          DCs were
                                                                                                                                                                                                                                                                                                                       associated with exosomal markers
                  Relative virus binding (p24gag)




                                                                                                                                          microscopy. (A) HIV
                                                                                                                   incubated with
                                                                                                                                          particles are present at the                                              0.5 µM                                                                                                                                                                                                                                            exosome-associated exocytosis.
                                                    100                          120                               virus (10ng p24gag)
                                                                                                                   for 2 h at 37oC or     cell surface                                                                                                                                                                                                    +                                                              +                                                                    - DCs maintain an immature phenotype 24h post-virus
                                                                                 100                                                      (white arrowhead),                                                                                                                                            Western Analysis                                1                                                 1
                                                    80                                                                                                                                                                                                                                                                                                 R n                                              R n
                                                                                                                                                                                                                                                                                                                                                                                              s
                                                                                                                   4oC, washed,                                                                                                                                                                                                                                                                                                                                                                 exposure.
                                                                                                                                                                                                                                                                                                                                             ed     -D ctio                                           -D ctio
                                                                                                                                                                                                                                                                                                                                                                           C

                                                                                                                                                                                                                                                             MVB
                                                                                                                                                                                                                                                                                                                                                                          D


                                                                                                                                          in macropinosomes (black                                                                            C                                                                                            ct
                                                                                                                   treated                                                                                                                                                                                                                        LAfra                                             LA a



                                                                                                                                                                                                                                                                                                                                                                                                                                   3T
                                                                                  80




                                                                                                                                                                                                                                                                                                                                                                                                                                     s
                                                                                                                                                                                                                                                                                                                                                                  d




                                                                                                                                                                                                                                                                                                                                         fe                                                                                                                                                   - Virus particles captured by DCs are trafficked to




                                                                                                                                                                                                                                                                                                                                                                                                                                 iru
                                                    60                                                                                                                                                                                                                                                                                                                                            H fr
                                                                                                                                                                                                                                                                                                                                                                te




                                                                                                                                                                                                                    MVB

                                                                                                                                                                                                                                                                                                                                                                                                                                 29
                                                                                                                                          arrowhead), and in coated                                                                                                                                                  MW
                                                                                                                   with/without 0.25%                                                                                                                                                                                                  in Cs α-Hup                                             α - up
                                                                                                                                                                                                                                                                                                                                                              ec




                                                                                                                                                                                                                                                                                                                                                                                                                               iv
                                                                                                                                                                                                                                                                                                                                                                                                                              EK
                                                                                  60                                                      endosomal comparments                                                                                                                                                     (kDa)             n D
                                                                                                                                                                                                                                                                                                                                                             f




                                                                                                                                                                                                                                                                                                                                                                                                                             La
                                                                                                                   trypsin for 10min at                                                                                                                                                                                              U          s                                                 s                                                                                             MVBs and are subsequently exocytosed.
                                                                                                                                                                                                                                                                                                                                                          In




                                                    40                                                                                                                                                                                                                                                                                                                                                                       H
                                                                                  40                               37oC to digest         (arrow). (B) and (C) HIV
                                                                                                                   surface-exposed        particles in MVB                                                                                                                                                            88                                                                                                                                                                      - Captured HIV-1 particles exocytosed by DCs are
                                                    20                            20                                                      (arrowhead).                                                                                                                                                                                                                                                                                                                                          associated with HLA-DR1, CD9, CD1b, and CD63
                                                     0
                                                              37oC       4oC
                                                                                   0
                                                                                            37oC          4oC
                                                                                                                   virus particles, and
                                                                                                                   lysed for analysis
                                                                                                                   of p24gag content
                                                                                                                   by ELISA
                                                                                                                                                                                                                                                                   0.5 µM


                                                                                                                                                                                                                                                                                                                      50
                                                                                                                                                                                                                                                                                                                                                                                                                                           }    CD63
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                positive vesicles, and remain infectious.
                                                                                                                                                                                                                     0.2 µM
                                                                       untreated            trypsin                                                                                                                                                                                                                         Lane      1            2          3                                 4                            5        6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                   2. Exosome-associated HIV-1 particles are more infectious
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      than cell-free virus stock on a per particle basis.
                                                                                                                                                                  HIV-1 particles are present within multivesicular                                                                                     CD63 is expressed in            immuno-isolated       HLA-DR1+
                                                          HIV-1 particles are endocytosed by DCs                                                                      bodies (MVB) following acute infection                                                                                                          vesicular fractions

								
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