BCA2/Rabring7 promotes tetherin-dependent HIV-1 restriction

Abstract

Host cell factors can either positively or negatively regulate the assembly and egress of HIV-1 particles from infected cells. Recent reports have identified a previously uncharacterized transmembrane protein, tetherin/CD317/BST-2, as a crucial host restriction factor that acts during a late budding step in HIV-1 replication by inhibiting viral particle release. Although tetherin has been shown to promote the retention of nascent viral particles on the host cell surface, the precise molecular mechanisms that occur during and after these tethering events remain largely unknown. We here report that a RING-type E3 ubiquitin ligase, BCA2 (Breast cancer-associated gene 2; also called Rabring7, ZNF364 or RNF115), is a novel tetherin-interacting host protein that facilitates the restriction of HIV-1 particle production in tetherin-positive cells. The expression of human BCA2 in "tetherin-positive" HeLa, but not in "tetherin-negative" HOS cells, resulted in a strong restriction of HIV-1 particle production. Upon the expression of tetherin in HOS cells, BCA2 was capable of inhibiting viral particle production as in HeLa cells. The targeted depletion of endogenous BCA2 by RNA interference (RNAi) in HeLa cells reduced the intracellular accumulation of viral particles, which were nevertheless retained on the plasma membrane. BCA2 was also found to facilitate the internalization of HIV-1 virions into CD63(+) intracellular vesicles leading to their lysosomal degradation. These results indicate that BCA2 accelerates the internalization and degradation of viral particles following their tethering to the cell surface and is a co-factor or enhancer for the tetherin-dependent restriction of HIV-1 release from infected cells.

Figure 1. BCA2 is a tetherin-interacting protein.

(A) GST pull-down analysis of 293T cells expressing either N-terminally Myc-tagged-tetherin (FL) or a mutant lacking the cytoplasmic tail domain (Δ1–20). Cell lysates were precipitated with either purified GST or GST-BCA2, followed by immunoblotting analysis with a Myc antibody to detect BCA2 binding (top panels). To control for the expression levels of GST, a Coomassie Brilliant Blue stained image is also shown (bottom panel). (B) Immunoprecipitation analysis of 293T cells expressing N-terminally HA-tagged-BCA2 together with either Myc-tetherin (FL) or its mutant (Δ1–20). Cell lysates were immunoprecipitated with HA antibodies, followed by immunoblotting analysis with either HA or Myc antibodies. (C) Immunoprecipitation analysis of 293T cells expressing Myc-tetherin together with HA-BCA2 (FL) or its deletion mutants (ΔRING, ΔC and ΔN). Asterisks indicate non-specific IgG bands. (D) Immunoprecipitation analysis of endogenous tetherin and BCA2. HeLa cell lysates were immunoprecipitated with either anti-tetherin monoclonal antibody or control mouse IgG followed by immunoblotting with the indicated antibodies. (E) Confocal microscopic analysis of HeLa cells expressing GFP-tagged tetherin and HA-BCA2 (scale bar, 10 µm). Cells were fixed, permeabilized and stained with HA antibodies (red) followed by confocal microscopy. The inset shows an expanded view of the area indicated by the white box in which an association of GFP-tetherin with HA-BCA2 at the plasma membrane is evident.

Figure 2. BCA2 inhibits HIV-1 particle production in cells expressing tetherin.

(A) Single-round virus release analysis was performed using the indicated cell types transfected with either 300 ng of pNL4–3 or pNL4–3ΔVpu along with the indicated amounts of pCMV-HA-BCA2. At 48 hours following transfection, cell supernatants were analyzed by p24 ELISA. Immunoblotting with a BCA2 antibody for both endogenous and HA-tagged BCA2 expression is shown in the bottom panels. (B) Tetherin-dependent effects of BCA2 on HIV-1 particle production. HOS cells were transiently transfected with 100 ng of pCMV-Myc-tetherin or its deletion mutant (Δ1–20, lacking the cytoplasmic tail) together with 300 ng of pNL4–3 and indicated amounts of pCMV-HA-BCA2, followed by p24 ELISA. (C) Vpu antagonizes the effects of BCA2 upon HIV-1 particle production. HeLa cells were transiently transfected with either the indicated amounts of Vpu or its deletion mutant (1–50, lacking a portion of the cytoplasmic domain) and 300 ng of pNL4–3ΔVpu was co-transfected with or without 300 ng of pCMV-HA-BCA2. After 48 hours, cell supernatants were analyzed by p24 ELISA. (D) Single-round virus release analysis was performed using HeLa cell transfected with 300 ng of pNL4–3 along with either pCMV-HA-BCA2 (WT), its RING finger-defective mutant (C228A/C231A) or tetherin-interacting motif defective BCA2 mutant (ΔC). At 48 hours following transfection, viral supernatants were analyzed by p24 ELISA. (E) Jurkat cells were transfected with either empty vector (EV) or pIRESpuro-BCA2 by electroporation and selected with puromycin for 24 hours. The stable expression of BCA2 on Jurkat cells was confirmed by BCA2 immunoblotting (top panel). Cells were then infected with either HIV-1_NL4–3 or HIV-1_NL4–3Δ_Vpu at a low multiplicity. Cell supernatants were harvested at the indicated time-points and subjected to p24 ELISA (bottom panel). (F) BCA2 reduces the level of cell-associated Gag protein. Immunoblotting analysis of the cell lysates described in (A) was performed. The numerical values below the blots indicate the Gag signal intensities determined by densitometry. The virus release efficiency was calculated as “Sup Gag per Total Gag (Cell Gag plus Sup Gag)”. (G) Pulse-chase analysis of HeLa cells transfected with pNL4–3ΔVpu together with either control vector or pCMV-HA-BCA2. Two days after transfection, the radiolabeled cells were harvested at the indicated times, and cell lysates were immunoprecipitated with anti-p24 antibody, and then analyzed by SDS-PAGE and autoradiography (left panel). The relative intensity of Gag bands was determined by densitometry (right panel).

Figure 3. BCA2 promotes the accumulation of HIV-1 virions in intracellular compartments.

Electron microscopic analysis of HeLa cells transfected with pNL4–3 and either control vector (A) or pCMV-HA-BCA2 (B), at a molar ratio of 1∶3. (Scale bars, 1 µm except where indicated).

Figure 4. BCA2 enhances HIV-1 virion trafficking to lysosomes.

(A) Confocal microscopic analysis of HeLa cells expressing pNL4–3 and either empty vector (top row) or pCMV-HA-BCA2 (bottom row), at a molar ratio of 1∶3 (scale bar, 10 µm). Note that these transfected cells also expressed Vpu. Cells were stained with anti-p24 (green) and anti-CD63 (red) antibodies and analyzed by confocal microscopy. (B) In the cultures described in (A), over 100 cells were analyzed for the subcellular localization of p24, which was either strongly evident at the plasma membrane (PM only), or intracellular accumulations as well as at the plasma membrane (Int + PM). The data are given as a percentage of the total cells. (C, D) HeLa cells transfected with 300 ng of pNL4–3 and either empty vector or pCMV-HA-BCA2, at a molar ratio of 1∶3, were treated with or without lysosomal inhibitors. Inhibitors were added to the medium 18 hours before harvesting. Cell lysates and supernatants were then analyzed by immunoblotting (C) and p24 ELISA (D). The final concentrations of leupeptin and NH₄Cl were 5 µg/ml and 2 mM, respectively. The Gag signal intensities and the virus release efficiency are shown below the blots, as in Fig. 2F.

Figure 5. The targeted depletion of BCA2 blocks the intracellular accumulation of viral particles.

(A) Single-round virus release analysis of HeLa cells treated with control siRNA or two different BCA2-targeted siRNA vectors for 24 hours, prior to transfection with pNL4–3 or pNL4–3ΔVpu. At 48 hours following transfection, cell supernatants were analyzed by p24 ELISA. Immunoblotting analysis with a BCA2 antibody to detect endogenous BCA2 expression is shown in the bottom panel. (B) Confocal microscopic analysis of HeLa cells treated with control siRNA (upper panels) or BCA2-targeted siRNA (lower panels), prior to transfection with pNL4–3ΔVpu (scale bar, 10 µm). After 48 hours following transfection, cells were fixed and immunostained with anti-p24 (green) and anti-CD63 (red) antibodies followed by confocal microscopy. (C) In the cultures described in (B), over 100 cells were analyzed for the subcellular localization of p24, as described in Fig. 4B. (D) HeLa cells were treated with control siRNA or BCA2-targeted siRNA for 24 hours, prior to transfection with pNL4–3 or pNL4–3ΔVpu as in (A). At 48 hours following transfection, cell supernatants were harvested (first supernatants) and cells were treated with either PBS or buffer containing the protease subtilisin (1 mg/ml) for 15 min, prior to re-harvesting of the cell supernatants (second supernatants). Both the first and second supernatants were then mixed and analyzed by p24 ELISA.