Severe acute respiratory syndrome coronavirus spike protein counteracts BST2-mediated restriction of virus-like particle release

Abstract

BST2/tetherin, an interferon-inducible antiviral factor, can block the cellular release of various enveloped viruses. We previously reported that human coronavirus 229E (HCoV-229E) infection can alleviate the BST2 tethering of HIV-1 virions by downregulating cell surface BST2, suggesting that coronaviruses are capable of encoding anti-BST2 factors. Here we report our new finding that severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) glycoprotein, similar to Vpu, is capable of antagonizing the BST2 tethering of SARS-CoV, HCoV-229E, and HIV-1 virus-like particles via BST2 downregulation. However, unlike Vpu (which downmodulates BST2 by means of proteasomal and lysosomal degradation pathways), BST2 downregulation is apparently mediated by SARS-CoV S through the lysosomal degradation pathway only. We found that SARS-CoV S colocalized with both BST2 and reduced cell surface BST2, suggesting an association between SARS-CoV S and BST2 that targets the lysosomal degradation pathway. According to one recent report, SARS-CoV ORF7a antagonizes BST2 by interfering with BST2 glycosylation¹ . Our data provide support for the proposal that SARS-CoV and other enveloped viruses are capable of evolving supplementary anti-BST2 factors in a manner that requires virus replication. Further experiments are required to determine whether the BST2-mediated restriction of authentic SARS-CoV virions is alleviated by the SARS-CoV spike protein.

Keywords: SARS coronavirus; coronavirus; human immunodeficiency virus; immune responses; innate immunity; virus classification.

Figure 1

BST2 inhibits coronavirus virus‐like particle (VLP) production. A‐D, 293T cells were cotransfected with SARS M and N (panel A), HA‐tagged HCoV‐229E M and N (panel B), or NL4.3delVpu (panel C) expression vectors, with (lane 2) or without a BST2 expression vector. Cells and supernatants were harvested and subjected to Western immunoblotting at 24 to 36 hours posttransfection. D, N proteins from medium or cell samples were quantified by scanning N band densities from immunoblots. Rations of N level in media to those in cells were determined for each sample and normalized to that of samples without BST2 coexpression. Data were derived from at least three independent experiments. *P < .05; **P < .01. (E‐G) 293T cells were transfected with SARS (panel E), HCoV‐229E (panel F), or HIV‐1 (panel G) VLP‐producing expression vectors as described for (A‐C). All tests were performed with 0.1 μg, 0.5 μg, or 2 μg of cotransfected BST2 expression plasmids (lanes 2, 3 and 4, respectively). H‐J, HeLa or BST2‐knockdown HeLa (HeLa/BST2‐) cells were transfected with the indicated SARS, HCoV‐229E, or HIV‐1 VLP‐producing plasmid. Cells and supernatants were harvested, prepared and subjected to Western immunoblotting at 48 to 72 hours posttransfection. Viral proteins were detected with anti‐SARS‐CoV M, anti‐HCoV‐229 N antiserum, or monoclonal antibodies against SARS‐CoV N, HA‐tagged HCoV‐229 M, or p24CA. BST2 was probed with a rabbit anti‐BST2 antibody. BST2, bone marrow stromal antigen 2; SARS‐CoV, severe acute respiratory syndrome coronavirus

Figure 2

Subtilisin treatment promotes virus‐like particle release. 293T cells were cotransfected with SARS CoV (A), HCoV‐229E M and N (B), or NL4.3delVpu (C) with or without a BST2 expression vector. Cells were split equally into two dish plates at 24 hours posttransfection. After an additional 4 hours, culture medium was removed, washed twice with PBS, and incubated with PBS containing subtilisin (1 mg/mL) for 10 minutes at 37℃. Supernatants were harvested and centrifuged through 20% sucrose cushions, and pellets and cell lysates were subjected to western immunoblotting. Viral proteins were probed as described in the Figure 1 caption. BST2, bone marrow stromal antigen 2; PBS, phosphate‐buffered saline; SARS‐CoV, severe acute respiratory syndrome coronavirus

Figure 3

Cysteine residues in the BST2 N‐terminal ectodomain are important for inhibiting VLP release. 293T cells were cotransfected with SARS M plus N (panel A), HA‐tagged HCoV‐229E M plus N (panel B), or NL4.3delVpu (panel C) plus a wild‐type or mutant BST2 expression vector (BST2/C3A) containing alanine substitutions for three cysteine residues in the BST2 ectodomain. Cells and supernatants were harvested and subjected to western immunoblotting at 24 to 36 hours posttransfection. BST2, bone marrow stromal antigen 2; VLP, virus‐like particle

Figure 4

SARS‐CoV S downregulates BST2. A, SARS‐CoV S reduced the inhibition of HIV‐1 VLP production by BST2. 293T cells were transfected with NL4.3delVpu alone (lane 1) or combined with BST2 (lane 2) plus Vpu (lane 3) or SARS‐CoV S (lane 4) expression vectors. Cells and supernatants were harvested and subjected to western immunoblotting at 24 to 36 hours posttransfection. B‐C, 293T cells were transfected with 100 ng of BST2 alone (lane 1) or BST2 plus 1 μg (lane 2) or 3 μg (lane 3) of a SARS‐CoV S (panel B) or Vpu expression vector (panel C). Cells were harvested and subjected to western immunoblotting at 24 hours posttransfection. BST2, bone marrow stromal antigen 2; SARS‐CoV, severe acute respiratory syndrome coronavirus; VLP, virus‐like particle

Figure 5

SARS‐CoV S downregulates BST2 via a lysosomal degradation pathway. 293T cells were transfected with BST2 (lane 1) or cotransfected with BST2 plus a Vpu (lanes 2‐4) or SARS‐CoV S (lanes 5‐7) expression vector. At 24 hours posttransfection, cells were either left untreated (lanes 1, 2, and 5), treated with 30 μM MG‐132 for 6 hours (lanes 3 and 6), or treated with 25 μM NH₄Cl (lanes 4 and 7) for 6 hours before harvesting and immunoblotting. BST2, bone marrow stromal antigen 2; SARS‐CoV, severe acute respiratory syndrome coronavirus

Figure 6

SARS‐CoV glycoprotein S colocalizes with BST2. 293 cells (panel A) were cotransfected with BST2 and a SRAS‐CoV expression vector. HeLa cells (panel B) were transfected with the SARS‐CoV S expression vector. At 24 to 36 hours posttransfection, cells were probed with an anti‐BST2 antibody before cell membrane permeabilization. SARS‐CoV S was probed with an anti‐S polyclonal antiserum. A rhodamine‐conjugated or FITC‐conjugated antirabbit or antimouse antibody served as a secondary antibody. C, SARS‐CoV S coexpression reduces BST2 cell surface expression. 293T cells were transfected with BST2 alone (middle panel) or together with a SARS‐CoV S expression vector (bottom panel). At 24 to 36 hours posttransfection, cells were fixed and probed with a rabbit anti‐BST2 antibody before the permeabilization of cell membranes, followed by a secondary FITC‐conjugated antirabbit antibody. Cells then were analyzed by flow cytometry. BST2, bone marrow stromal antigen 2; SARS‐CoV, severe acute respiratory syndrome coronavirus