Fig 2: Innate sensing by BST2 is dispensable for its role in HIV-1 spreading.(A) BST2 surface levels on Jurkat E6.1 BST2-/- knockout cells reconstituted with variants of human BST2. (B and C) Both signaling-competent and signaling-defective versions of human BST2 fully rescue WT (Vpu+/Nef+) HIV-1NL4-3 replication in the knockout cells, as examined by Western blotting of cell lysates with anti-CA on day 10 after infection with 0.2 ng p24/ml (B) and in parallel by p24 ELISA of culture supernatants (C). (D) BST2 surface levels on Jurkat E6.1 BST2-/- knockout cells reconstituted with mouse BST2. (E and F) Mouse BST2 partially restores WT HIV-1NL4-3 replication in the knockout cells, as examined by monitoring cell-associated Gag levels (E) and p24 antigen release (F). PE, phycoerythrin.

Fig 3: HIV-1 propagation is highly dependent on the Vpu target BST2.(A) Expression of BST2 on parental MOLT-3 cells and on FACS-sorted subpopulations. The BST2+/+ (TS1) and BST2-/- (TS1) subpopulations were obtained by sorting after one round of gene editing with sgRNA TS1, and the BST2-/- (TS1/2) subpopulation was obtained by subjecting BST2-/- (TS1) cells to a second round of editing with sgRNA TS2, followed by a second round of sorting. Open histograms represent staining with anti-BST2 mAb; gray-shaded histograms represent the isotype control. (B and C) Virus growth curves showing that the propagation of WT (Vpu+/Nef+) HIV-1NL4-3 in the BST2-/- (TS1) subpopulation is highly impaired (B) and is even more impaired in the twice-sorted BST2-/- (TS1/2) subpopulation (C), which lacks detectable surface BST2. All subpopulations were infected with 0.2 ng p24/ml, and virus release was monitored by p24 enzyme-linked immunosorbent assay (ELISA). (D) Virus propagation in the same cultures monitored by comparing Gag expression levels in the infected cells at different days after infection by Western blotting with anti-CA. pI, post infection. (E) Expression of BST2 on FACS-sorted Jurkat E6.1 subpopulations. The BST2+/+ (TS1) and BST2-/- (TS1) subpopulations were obtained after gene editing with an sgRNA (TS1) that targets the BST2 gene, whereas two sgRNAs (TS3 and TS4) that target sites flanking the BST2 gene were used simultaneously to generate the BST2+/+ (TS3+4) and BST2-/- (TS3+4) subpopulations. (F) Propagation of WT HIV-1NL4-3 in Jurkat E6.1 subpopulations monitored by p24 ELISA after infection with 0.2 ng p24/ml. (G and H) Propagation of WT HIV-1NL4-3 in the BST2+/+ (TS3+4) and BST2-/- (TS3+4) subpopulations after infection with 0.2 or 2 ng p24/ml, monitored in parallel by p24 ELISA (G) and by Western blotting of cell lysates with anti-CA (H).

Fig 4: The ability of art-BST2 to support HIV-1 propagation depends on its virus-tethering activity.(A) Schematic illustration of art-BST2 mutants that lack the N-proximal transmembrane domain (art-BST2?TM), the extracellular coiled coil (art-BST2?CC), or the C-terminal GPI anchor (art-BST2?GPI). All mutants retain the HA tag in the art-BST2 ectodomain. (B) Virus growth curves showing that the two membrane anchors and the coiled coil of art-BST2 are all essential for its ability to rescue WT (Vpu+/Nef+) HIV-1NL4-3 replication in BST2-/- knockout cells. BST2+/+ (TS3+4) and BST2-/- (TS3+4) Jurkat E6.1 subpopulations stably transduced with empty vector or vectors encoding the indicated versions of art-BST2 were infected with 0.2 ng p24/ml, and virus replication was examined by monitoring p24 antigen release. (C) Comparison of cell-associated Gag levels in the same cultures by Western blotting with anti-CA. (D) Surface expression of WT and mutant versions of art-BST2 on stably transduced Jurkat E6.1 BST2-/- knockout cells examined by flow cytometry with anti-HA.

Fig 5: BST2 is dispensable for infectability but required for HIV-1 spreading.(A) Dot plots showing that BST2+/+ and BST2-/- Jurkat E6.1/ZsGreen subpopulations are equally susceptible to single-cycle infection with cell-free WT (Vpu+/Nef+) HIV-1NL4-3. The entry inhibitor AMD3100 was added at 0 or 12 hours after infection with WT HIV-1NL4-3 at a high multiplicity of infection (MOI). Infected (ZsGreen+) cells were quantified by flow cytometry. (B) In the same cells, BST2 is essential for spreading infections. BST2+/+ and BST2-/- Jurkat E6.1/ZsGreen subpopulations were infected with a low amount of cell-free WT HIV-1NL4-3 (2 ng p24/ml), and after 10 days infected (ZsGreen+) cells were detected by fluorescence microscopy. FSC, forward scatter.