BCA2/Rabring7 targets HIV-1 Gag for lysosomal degradation in a tetherin-independent manner

Abstract

BCA2 (Rabring7, RNF115 or ZNF364) is a RING-finger E3 ubiquitin ligase that was identified as a co-factor in the restriction imposed by tetherin/BST2 on HIV-1. Contrary to the current model, in which BCA2 lacks antiviral activity in the absence of tetherin, we found that BCA2 possesses tetherin-independent antiviral activity. Here we show that the N-terminus of BCA2 physically interacts with the Matrix region of HIV-1 and other retroviral Gag proteins and promotes their ubiquitination, redistribution to endo-lysosomal compartments and, ultimately, lysosomal degradation. The targeted depletion of BCA2 in tetherin-expressing and tetherin-deficient cells results in a significant increase in virus release and replication, indicating that endogenous BCA2 possesses antiviral activity. Therefore, these results indicate that BCA2 functions as an antiviral factor that targets HIV-1 Gag for degradation, impairing virus assembly and release.

Figure 1. BCA2 has tetherin-independent antiviral activity.

The antiviral activity of BCA2 was tested by co-transfecting 293T cells in duplicate with HIV-1 NL4-3, HIV-1 NL4-3 Δvpu, SIV_mac239 or SIV_mac239 Δnef proviral DNA in the presence of constructs coding for human or rhesus tetherin (hBST2, rBST2), HA-BCA2 or empty vector. Differences in DNA concentrations were offset by the addition of empty vector, and results were confirmed in three additional independent experiments. (A) Virus release for HIV-1 was measured by p24 antigen-capture ELISA 48 hours post-transfection. (B) The whole cell lysates (WCL) and virions generated from these transfections were analyzed by western blot to assess the expression levels of tetherin, HA-BCA2 and viral proteins (Nef, Vpu, Gag, CA: p24). (C) Virus release for SIV was assessed by p27 antigen-capture ELISA. (D) The expression levels of tetherin, HA-BCA2 and viral proteins were also assessed by western blot (Nef, Gag, CA: p27). (E) 293T cells were co-transfected with equal amounts of HIV-1 or SIV proviral DNA and increasing concentrations of expression vectors coding for HA-BCA2 or ΔRing BCA2. Differences in DNA concentration were offset by adding empty vector. Virus release was determined as described above, and expressed as the percentage of maximal virus release in the absence of HA-BCA2 or the ΔRing BCA2 mutant. To determine the infectivity of influenza H1N1, 293T cells were infected with 1 ml of H1N1 virions produced from HA-BCA2-expressing cells. Infectivity was determined 24 hours post-infection by measuring the amount of influenza hemagglutinin protein (HA) protein present on the cell surface. Similar to panels B and D, whole cell lysates and virions generated from transfections with HA-BCA2 (F) and ΔRing BCA2 (G) were analyzed by western blot. Error bars represent standard deviation of independent experiments.

Figure 2. The E3 ligase activity of BCA2 is required for antiviral activity.

(A) Schematic representation of BCA2 with the N-terminal BCA2 Zinc-finger domain (BZF), the AKT-phosphorylation sites, and the C-terminal RING-finger domain. Key residues are indicated. (B) Virus release assays for HIV-1 NL4-3, SIV_mac239 and Mo-MLV were performed from cells expressing wild-type HA-BCA2 or the indicated BCA2 mutants, and expressed as the percentage of maximal release, as previously described. The whole cell lysates (WCL) and the virions produced were also analyzed by western blot to assess HA-BCA2, Gag, Nef, CA and β-actin expression. Red asterisk indicates MW of 37 KDa. (C) The tetherin-independent and tetherin-dependent antiviral activity of BCA2 was examined in a virus release assay from parental 293T and 293T cells stably expressing tetherin. Results were also corroborated by western blot of the whole cell lysates (WCL) and pelleted virions. Error bars represent standard deviation of independent experiments.

Figure 3. BCA2 induces the ubiquitination of retroviral Gag proteins.

Retroviral Gag proteins were tested for BCA2-induced ubiquitination by co-transfecting 293T cells with vectors expressing codon-optimized HIV-1 NL4-3, SIV_mac239 and Mo-MLV Gag proteins along with an expression vector coding for HA-BCA2 or empty vector. (A) Cell lysates were incubated with a polyubiquitin affinity resin and the bound fraction was eluted and analyzed by western blot. Membranes were probed with antibodies specific for p55/p24, MLV p30 and HA. Whole cell lysates were set aside for western blot analyses. (B) The BCA2-induced ubiquitination of Gag-GFP as well as native Gag was analyzed in side-by-side experiments. 293T cells were co-transfected in duplicate with each Gag construct, HA-BCA2, ΔRing BCA2 or empty vector. Cell lysates were set aside for regular western blotting, and the rest of the lysates were immunoprecipitated with an antibody anti-CA. Membranes were developed with an anti-Ubiquitin (Ub) antibody. (C) The ubiquitination of HIV-1 Gag, or HIV-1 Gag mutants, in cells expressing HA-BCA2 was also analyzed by immunoprecipitation. Cell lysates were set aside for western blot analyses and the rest of the samples were immunoprecipitated with an anti-CA specific antibody. Membranes were probed with an anti-Ubiquitin antibody. Similarly, the ubiquitination levels of HA-BCA2 were also determined. In this case, samples were immunoprecipitated using an anti-HA antibody. Results were confirmed in two additional independent assays. (D) A similar approach was used to analyze the ubiquitination levels of SIV Gag, or SIV Gag mutants, in the presence and absence of HA-BCA2. (E) Schematic representation of the putative lysine residues susceptible to become ubiquitinated by BCA2 in HIV-1 and SIV Gag (red). IP: immunoprecipitation. IB: immunoblot. WCL: whole cell lysate. V: empty vector.

Figure 4. The N-terminus of BCA2 interacts with the Matrix region of Gag.

(A) To investigate if retroviral Gag proteins interact with BCA2, 293T cells were co-transfected with HIV-1, SIV and Mo-MLV Gag constructs and either empty vector or a vector encoding HA-BCA2. Cell lysates were immunoprecipitated with a CA-specific antibody and membranes were probed with an anti-HA antibody. Results obtained from these assays were corroborated independently twice. (B) Schematic representation of the Gag and HA-BCA2 deleted constructs used. Similar to panel A, HIV (C) and SIV (D) Gag deleted mutants were tested for an interaction with HA-BCA2 by co-immunoprecipitation. Likewise, HA-BCA2 deleted mutants were tested for an interaction with HIV (E) and SIV (F) Gag proteins by co-immunoprecipitation. In this case, lysates were immunoprecipitated with an anti-HA antibody and western blot membranes were probed with a GFP-specific antibody. Whole cell lysates (WCL) were set aside to check the input levels of these proteins and β-actin. IP: immunoprecipitation. V: empty vector.

Figure 5. BCA2 promotes the lysosomal degradation of HIV-1 and SIV Gag.

To determine if BCA2 promotes the proteasomal or lysosomal degradation of retroviral Gag proteins, virus release assays for HIV-1 and SIV were performed in the presence of proteasomal inhibitors (A-B) and lysosomal inhibitors (C-D). Virus release was measured by HIV-1 p24 or SIV p27 antigen-capture ELISA and expressed as the percentage of maximal virus release in the absence of HA-BCA2. The cell lysates (WCL) and virions derived from these transfections were analyzed by western blot. Clasto: clasto-Lactacystin β-lactone. Chlor: chloroquine. Error bars represent standard deviation of independent experiments.

Figure 6. The targeted depletion of endogenous BCA2 results in increased virus release and replication.

Endogenous BCA2 was depleted by shRNA from 293T cells (A) and HOS cells (B) by transient transfection. Cells were subsequently transfected with HIV-1 NL4-3 or SIV_mac239 proviral DNA, and virus release was measured 48 hours later. Depletion of endogenous BCA2 was also achieved in CD4⁺ T cells by transduction. Next, virus replication was assessed for HIV-1 in Jurkat cells (C) and for SIV in 221 T cells (D) by HIV-1 p24 and SIV p27 antigen-capture ELISA, respectively, at selected time points. The depletion of endogenous BCA2 was confirmed by western blot for each of these cell lines, by comparing the levels of endogenous BCA2 in the whole cell lysate (WCL) to those of β-actin. Error bars represent standard deviation of independent experiments.

Figure 7. BCA2 leads to the accumulation of Gag proteins within intracellular compartments.

The effects of BCA2 on Gag distribution were investigated by confocal microscopy. 293T cells were co-transfected with constructs coding for HA-BCA2 and HIV-1 Gag-GFP, SIV Gag-GFP or empty vector. Similar assays were performed in the presence of HIV-1 NL4-3 and SIV_mac239 proviral DNA. Cells were stained for HA-BCA2 (red), Gag-GFP or Capsid (p24 or p27) (green) and the nuclei (blue). (A) Cellular distribution of HA-BCA2 in Gag-deficient cells. (B) Cellular distribution of HIV-1 Gag-GFP, SIV Gag-GFP (left panels) and HIV-1 p24 or SIV p27 (right panels) in HA-BCA2-deficient cells. (C) Cellular distribution of HIV-1 Gag-GFP and HIV-1 p24 in cells expressing HA-BCA2. (D) Cellular distribution of SIV Gag-GFP and SIV p27 in HA-BCA2-expressing cells. The white scale bar corresponds to 10 µm for images with more than one cell, and 7.5 µm for images with single cells.

Figure 8. BCA2 re-distributes HIV-1 Gag, but not HIV-1 Gag G₂A, to endo-lysosomal compartments.

(A) Distribution of the HIV-1 Gag-G₂A-GFP mutant in HA-BCA2⁻ (top panel) and HA-BCA2⁺ (bottom panels) cells. (B-C) The intracellular localization of native HIV-1 Gag in the presence of HA-BCA2 was determined by staining cells for p24 (green), HA-BCA2 (blue) and either CD63, or LAMP1 (red). The white scale bar corresponds to 10 µm for images with more than one cell, and 7.5 µm for images with single cells.