Abstract
The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS- CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.
Author Summary Since it was identified in 2019, SARS-CoV-2 has displayed voracious transmissibility which has resulted in rapid spread of the virus and a global pandemic. SARS-CoV-2 is a member of the Coronaviridae family whose members are encapsulated by a host-derived protective membrane shell. Whilst the viral envelope may provide protection for the virus, it also provides an opportunity for the host cell to restrict the virus and stop it spreading. The anti-viral restriction factor, tetherin, acts to crosslink viruses to the surface of infected cells and prevent their spread to uninfected cells. Here, we demonstrate that SARS-CoV-2 undergoes viral restriction by tetherin, and that SARS-CoV-2 moves tetherin away from the site of Coronavirus budding to enhance its ability to escape and infect naïve cells. Tetherin depletion from cells enhanced SARS-CoV-2 viral release and increased propagation of the virus. We found that the SARS-CoV-2 protein, ORF3a, redirects tetherin away from the biosynthetic organelles where tetherin would become incorporated to newly forming SARS-CoV-2 virions – and instead relocalises tetherin to late endocytic organelles. We also found that SARS-CoV-2 Spike downregulates tetherin. These two mechanisms, in addition to the well described antagonism of interferon and subsequent ISGs highlight the multiple mechanisms by which SARS-CoV-2 abrogates tetherin function. Our study provides new insights into how SARS-CoV-2 subverts human antiviral responses and escapes from infected cells.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Abbreviations ACE2, angiotensin converting enzyme 2, Bst2, bone marrow stromal antigen 2, DMV, double membrane vesicle, ERGIC, endoplasmic reticulum-Golgi intermediate compartments, GPI, glycosylphosphatidylinositol, IFN, interferon, LAMP1, Lysosomal-associated membrane protein 1, ORF, open reading frame, SARS, severe acute respiratory syndrome.
This manuscript has been updated to incorporate new data. We now include additional data demonstrating a role for SARS-CoV-2 ORF3a in tetherin antagonism. Expression of ORF3a causes a shift in the steady-state distribution of tetherin, moving it away from the biosynthetic pathway and towards the lysosomal pathway. We demonstrate that the ORF3a-mediated relocalisation of tetherin occurs due to defective retrograde traffic. Although ORF3a does not reduce tetherin protein levels, the redistribution of tetherin away from the SARS-CoV-2 budding organelles reduces VLP retention. These data have no impact upon our original findings, and we reproducibly observe tetherin downregulation by SARS-CoV-2 Spike - allbeit to an insufficient degree to change VLP release.
