Severe acute respiratory syndrome coronavirus spike protein counteracts BST2‐mediated restriction of virus‐like particle release
First published: 14 June 2019
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.
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.
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BST2/tetherin inhibits the release of various enveloped viruses.
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SARS‐CoV S antagonizes the BST2 tethering of human coronavirus and HIV‐1 virus‐like particles.
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SARS‐CoV S colocalizes with BST2 and reduces cell surface BST2.
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SARS‐CoV S downregulates BST2 through lysosomal degradation pathway.
One research team has proposed that BST2 inhibits virus release by tethering nascent virions to cell surfaces via the N‐terminal transmembrane domain and C‐terminal GPI anchor.14
Most BST2‐restricted enveloped viruses bud directly from cell surfaces, but a small number of enveloped viruses (eg, herpesviruses) are subject to BST2‐related restrictions even though their final envelopment entails membranes from TGN and/or endosomal compartments and egression via exocytosis.15, 16
In a previous study we reported that the human coronavirus 229E (HCoV‐229E), whose assembly and budding occurs at the ER‐Golgi intermediate compartment and whose virions are released via vesicle exocytosis,17-19 is also subject to BST2 inhibition. Results from electron microscopy analyses indicate the presence of HCoV‐229E virions on cell surfaces or on the membranes of intracellular vesicles that tend to cluster with BST2. This suggests the BST2‐triggered tethering of budding virions to vesicle membranes that remain on cell surfaces at the plasma membrane after exocytosis.17
BST2 has been described as moderately restricting the release of the hepatitis C virus, whose assembly takes place in the ER and whose release from cells via secretory pathways occurs in a manner similar to that of coronaviruses.20, 21
Combined, these data support the assumption that enveloped virus budding and release occurring at the plasma membrane or in an intracellular compartment is subject to BST2 blocking. BST2 is a component of innate immune response in the form of restricted enveloped virion release, and many viruses have evolved specific antagonists to counteract BST2 antiviral activity: HIV‐1 Vpu, HIV‐2 Env, simian immunodeficiency virus (SIV) Nef and Env, Ebola and Sendai virus GP, Kaposi's sarcoma‐associated herpesvirus K5, and influenza virus neuraminidase are all capable of antagonizing BST2.2-5, 9, 12, 13, 15, 22
Since some of these anti‐BST2 viral factors are viral envelope glycoproteins, there is speculation that SARS‐CoV spike glycoprotein may possess the property to counteract the BST2 blocking of virus release. Our work is built in part on an earlier finding by another research team that the ORF7a accessory protein (encoded by SARS‐CoV) inhibits the BST2 tethering of virions.23
We also found that the SARS‐CoV spike (S) protein is capable of downmodulating BST2, thus mitigating the BST2‐mediated restriction of virus‐like particle (VLP) release, and suggesting that SARS‐CoV and other enveloped viruses are capable of evolving additional anti‐BST2 factors.
Combined, the data suggest that both exogenous and endogenous BST2 are capable of inhibiting SARS‐CoV and HCoV‐229E VLP release.
BST2 forms stable cysteine‐linked dimers. Blocking BST2 dimerization by replacing cysteines C53, C63, and C91 with alanine in the BST2 ectodomain, in turn, blocks the BST2 inhibition of HIV‐1 release, suggesting that such dimerization is required for virion release blocking.25
To test whether BST2 dimerization is also required for restricting coronavirus VLP release, M and N proteins were coexpressed with a dimerization‐defective BST2/C3A mutant containing alanine substitutions for C53, C63, and C91. Results indicate that wild‐type BST2 was capable of inhibiting SARS‐CoV, HCoV‐229E, and HIV‐1 VLP production, but BST2/C3A was not (Figures 3A‐C, lane 2 vs. lane 3), further suggesting that BST2 dimerization is required to inhibit coronavirus VLP release.
SARS‐CoV spike (S) alleviates BST2 restriction of HIV‐1 release by downregulating BST2
As stated above, several viral membrane glycoproteins
such as HIV‐2 and SIV Env, as well as Ebola and Sendai virus GP
proteins, exert counteractive effects on BST2. We, therefore, attempted
to identify anti‐BST2 activity associated with the SARS‐CoV spike (S)
protein. Since BST2 is known to restrict HIV‐1 release in the absence of
Vpu, we performed tests to determine whether SARS‐CoV S counteracts
BST2 and therefore supports HIV‐1 release. As shown in the upper panel
of Figure 4A
(lane 2 vs. lanes 3 and 4), the inhibitory effect of BST2 on
NL4.3delVpu virion release decreased in the presence of either SARS‐CoV S
or Vpu in step with reduced BST2 expression (Figure 4A,
middle panel, lanes 2‐4), suggesting that SARS‐CoV S is capable of
promoting the release of HIV‐1 virus particles from cells via BST2
downregulation. Additional experiments confirmed that SARS‐CoV S, like
Vpu, is capable of reducing BST2 expression in a dose‐dependent manner
(Figures 4B and C). Combined, the data suggest that SARS‐CoV S may counteract the BST2‐mediated restriction of VLP release.
4 DISCUSSION
BST2 is capable of inhibiting SARS‐CoV and HCoV‐229E VLP release (Figure 1).
Since the BST2 inhibition of HIV‐1 release via virion tethering at cell
surfaces is well documented, we used a Vpu‐deficient HIV‐1
virus‐producing vector (NL4.3delVpu) as a control in our experiments. As
shown in Figure 2,
coronavirus VLPs (similar to those of HIV‐1) were tethered to cell
surfaces by BST2, and BST2 inhibited SARS‐CoV and HCoV‐229E VLP release
in a BST2 dimerization‐dependent manner, similar to HIV‐1 (Figure 3). Further, in the same manner, as Vpu, SARS‐CoV S facilitated HIV‐1 release via BST2 downregulation (Figure 4).We determined that Vpu downmodulated BST2 via proteasomal and lysosomal degradation pathways and that the predominant lysosomal pathway was mediated by SARS‐CoV S (Figure 5).
Our confocal microscopy observations suggest that SARS‐CoV S colocalized with BST2 at HeLa cell surfaces (Figure 6).
SARS‐CoV S likely binds to BST2, after which it serves as a target for lysosomal degradation. Vpu is capable of trapping BST2 intracellularly and preventing its recycling back into the plasma membrane.29, 35, 37, 42, 43
Whether SARS‐CoV S similarly counteracts BST2 requires further investigation.
The transmembrane protein SARS‐CoV ORF7a has been shown to counteract BST2 tethering by interfering with BST2 glycosylation.23, 44
In addition to the likelihood that BST2‐associated restriction of SARS‐CoV virion release is mitigated by SARS‐CoV S, it is possible that a number of enveloped viruses have developed supplementary anti‐BST2 factors over time—note that in addition to Vpu, HIV‐1 Nef is capable of overcoming BST2 restrictions on virus release under certain conditions.45
SIV Nef7 and Env2 are capable of antagonizing BST2, and influenza neuraminidase12 and M246 proteins both possess anti‐BST2 capabilities.
Some researchers have suggested that influenza and/or Ebola VLP release, but not virion release, is inhibited by BST2.11, 47
Due to biosafety requirements, we are currently unable to perform tests to determine whether SARS‐CoV S is capable of overcoming BST2 restrictions on SARS‐CoV virion release.
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