Supplementary Materials

Retroviruses use CD169-mediated trans-infection of permissive lymphocytes to establish infection

Xaver Sewald, Mark S. Ladinsky, Pradeep D. Uchil, Jagadish Beloor, Ruoxi Pi, Christin Herrmann, Nasim Motamedi, Thomas T. Murooka, Michael A. Brehm, Dale L. Greiner, Leonard D. Shultz, Thorsten R. Mempel, Pamela J. Bjorkman, Priti Kumar, Walther Mothes

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S19
  • Captions for Movies S1 to S14

Images, Video, and Other Other Media

Movie S1
MLV Gag-GFP is captured at the pLN subcapsular sinus (SCS) floor. 3D rendering of time-lapse intravital 2-Photon laser scanning microscopy (2P-LSM) showing the arrival of MLV Gag-GFP (green) at the SCS floor of a pLN that is outlined by the collagen capsule (blue). The timelapse was taken every 30 sec in a 3D space of 400 x 400 x 60 μm with 3 μm z-spacing over a period of 60 min after s.c. injection into a C57BL/6 mouse.
Movie S2
Capture of HIV Gag-GFP at the pLN SCS floor. Time-lapse movie as for movie S1 showing the arrival of HIV Gag-GFP (green) at the SCS floor of a pLN (collagen, blue). The timelapse video was taken over a period of 40 min following the s.c. MLV injection into a C57BL/6 mouse.
Movie S3
CD169+ macrophages capture MLV particles at the pLN SCS floor. A z-stack of PFA-fixed pLN 1 h after s.c. injection with MLV Gag-GFP (green). The movie starts at the top of the pLN where the collagen capsule is visible in blue. CD169+ macrophages were stained by injecting CF568-labeled antibodies to CD169 (red) 30 min after virus. Images of pLN explants were taken at a depth of 0-60 μm and a 3D space of 450 x 450 x 60 μm with 1 μm z-spacing.
Movie S4
MLV does not infect naïve B cells in the pLN. An extended focus projection of timelapse intravital 2P-LSM of a pLN containing naïve RFP+ B cells (red) 2 days after s.c. infection with MLV reporter virus (MLV LTR-GFP). MLV-infected cells express GFP (green). RFP+ naïve B cells were adoptively transferred 18 h before MLV infection. Note that none of the naïve RFP+ B cells are green and hence are not infected by MLV. Images were taken every 30 sec over a period of 45 min in a 3D space of 300 x 300 x 60 μm with 3 μm z-spacing.
Movie S5
B-1 cells sample the SCS space with MLV-laden macrophages. 3D rendering of timelapse intravital 2P-LSM at the pLN of a C57BL/6 mouse carrying adoptively transferred RFP+ B-1 cells (red) 2 min after s.c. injection of fluorescently labeled MLV Gag-GFP particles (green). Collagen of pLN capsule is shown in blue. Images were taken every 30 sec in a 3D space of 400 x 400 x 60 μm with 3 μm z-spacing.
Movie S6
B-1 cells establish synaptic contact with MLV-laden SCS macrophages. 3D rendering of time-lapse intravital 2P-LSM at the pLN of a C57BL/6 mouse as adoptively transferred RFP+ B-1 cells (red) establish synaptic contact with MLV Gag-GFP (green)-laden SCS macrophages 3 h after s.c. injection. Collagen of pLN capsule in blue. Arrows indicate two examples of static B-1 cells in contact with SCS macrophages. Images were taken every 30 sec over a period of 45 min in a 3D space of 200 x 200 x 60 μm with 3 μm z-spacing.
Movie S7
B-1 cells form stable long-lived synaptic contact with MLV-laden SCS macrophages. 3D rendering of time-lapse intravital 2P-LSM at pLN of mice demonstrating long-lived contact (30 min) of adoptively transferred RFP+ B-1 cells (red) with MLV Gag-GFP (green)-laden SCS macrophages at the pLN SCS 3 h after s.c. injection of virus. Images were taken every 30 sec over 30 min at a 3D space of 110 x 110 x 60 μm with 3 μm z-spacing.
Movie S8
B-1 cell carrying MLV Gag-GFP material at its uropod following a long-lived interaction with a SCS macrophage. An extended focus projection of time-lapse intravital 2P-LSM in which a RFP+ B-1 cell (red) first interacts with a macrophage and all MLV Gag-GFP (green) 25 material is concentrated at the cell-cell interface. When the B-1 cell begins to migrate, MLV Gag- GFP (green) material localizes to its trailing uropod. The pLN SCS region was imaged 3 h after s.c. virus injection. Top view (x-y) is shown at the left and sideview (x-z) at the right. Collagen of pLN capsule is shown in blue. Images were taken every 30 sec over a period of 18 min at a 3D space of 90 x 90 x 60 μm (top view) and 12 x 25 x 60 μm (side view) with 3 μm z-spacing.
Movie S9
Migrating B-1 cells carry MLV particles at its uropod. A time-lapse intravital 2PLSM for a similar event as in movie S8. The movie lasts over 25 min in a 3D space of 125 x 125 x 60 μm with 3 μm z-spacing.
Movie S10
Electron tomography of a MLV-laden SCS macrophage. Montaged tomographic reconstruction of a SCS macrophage in a mouse pLN 1 h after s.c. injection of MLV. Large numbers of virus particles are found at the surfaces and in numerous plasma membrane invaginations of the macrophage. Nearly all virus particles are mature. The size bar corresponds to 200 nm.
Movie S11
MLV-infected B-1 cells form stable virological synapses in vivo. An extended focus projection of intravital time-lapse 2P-LSM of pLN 2 days after s.c. infection with replication competent MLV labeled with Gag-GFP. MLV-infected cells express Gag-GFP (green). RFP+ B-1 cells (red) were adoptively transferred s.c. 18-24 h before MLV infection. Note that incoming MLV Gag-GFP can infect adoptively transferred B-1 cells (red, green) as well as endogenous unlabeled B- 1 cells (green). Two regions of interest (ROI1, ROI2) are presented to the right for examples of stable virological synapses with polarized Gag-GFP accumulation in MLV-infected RFP+ B-1 cells. Images were taken every 30 sec over a period of 30 min in a 3D space of 280 x 280 x 60 μm with 3 μm zspacing. ROIs are 90 x 90 x 60 μm (ROI1) and 70 x 70 x 60 μm (ROI2) in size.
Movie S12
Stages of MLV spread in vivo via cell-cell contacts. Extended focus projection of intravital 2P-LSM of a pLN 2 days after s.c. infection with replication competent MLV labeled with Gag-GFP. RFP+ B-1 cells (red) were adoptively transferred through s.c. injection 18-24 h before MLV infection. The left panel shows a MLV-infected cell (green) forming a stable contact with an uninfected B-1 cell (red). The middle panel shows the polarization of Gag-GFP towards the cell-cell interface with a B-1 cell (red). The right panel shows transfer of MLV Gag-GFP material to an unlabeled non-infected endogenous cell. Images were taken every 30 sec over a period of 25-30 min. The 3D space of left, middle and right panel are 80 x 80 x 40 μm, 70 x 70 x 9 μm and 80 x 80 x 45 μm respectively, with 2 μm, 3 μm and 3 μm z-spacing respectively.
Movie S13
Electron tomography of a virological synapse. Tomographic reconstruction of viruscontaining membrane protrusions in a pLN 2 days post s.c. infection with MLV. This movie corresponds to Figure 3F. The infected donor cell is located to the upper right, outside of the field of view with the uropod extending a considerable distance and curving around other nearby cells. Viruses associated with these protrusions are exclusively immature suggesting that maturation occurs after entry (38). The size bar corresponds to 0.5 μm.
Movie S14
Electron tomography of a MLV containing uropod complex at a virological synapse. Serial-section tomographic reconstruction of a large, virus containing membrane protrusion in a pLN 2 days post s.c. infection with MLV. This movie corresponds to fig. S18. The infected donor cell is at the lower left and the virus containing protrusion extends outward to contact a cell at the upper right. All of the MLV particles within the reconstruction are immature. The size bar corresponds to 0.5 μm.