Figure 3

MDMs were infected with VSV-G-pseudotyped HIV-1 NL4-3. Cells were harvested at day 8, fixed, embedded, and sectioned. Sections were etched with H₂O₂ and labeled with 6 nm gold beads using anti-tetherin antisera using methods previously established (Hammonds et al., 2010). (A) Multiple nanogold beads between particles at low power within a VCC, bar = 200 nm. Arrows added to point out beads. (B) Higher magnification view of left half of A, bar = 100nm. (C) Higher magnification view of upper right portion of A, bar = 100 nm. (D) Extended tether and beads between particles, bar = 100nm. (E) Tetherin at base of particles, between limiting membrane and particles within intracellular compartment in macrophages. Bar = 100nm. (F) Tetherin immunolabeling at base of particle and between particles within intracellular compartment in macrophages. Bar = 100nm. (G) Tetherin linking particle to the limiting membrane of a VCC, bar = 100nm. (H, I) Additional views of tetherin labeling within VCCs, on surface of virions and between virions. (J) VCC, control section employing pre-immune serum and immunogold labeling otherwise identical to that in A-I. Bar = 500nm. (K) Detail of same VCC as in (J) with control (preimmune) labeling, bar = 100nm. (L) Gold particles found within VCCs of HIV-infected macrophages, mean ± SD, using specific anti-tetherin sera (black) or preimmune sera (gray). Twenty consecutive VCCs with virions were assessed for each label, and normalized by total number of visualized particles. See Figure S3 for assessment of macrophage purity by flow cytometry.