Life Sciences, Lausanne-Switzerland have in a new study found that acylation of spike of SARS-CoV-2 enhances infectivity and also fusion capacity.
The study team found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi.
The 4 key findings of the study were:
- SARS-CoV-2 spike proteins are massively S-acylated during infection
- ZDHHC20 is the dominant host enzyme in this process
- S-acylation of spike generates cholesterol-rich lipid domains within viral envelopes
- S-acylation and lipid biosynthesis pathways promote SARS-CoV-2 infectivity
- The study findings were published in the peer reviewed journal: Developmental Cell (Scient Direct By Elsevier)
β-coronaviruses (CoVs) are positive-stranded RNA enveloped viruses that infect a wide range of hosts (V’kovski et al., 2021). Human-tropic species can cause from mild respiratory symptoms to life-threatening forms of atypical pneumonia termed severe acute respiratory syndrome (SARS) (V’kovski et al., 2021; Wang et al., 2020), such as COVID-19 caused by SARS-CoV-2 (SARS coronavirus 2) (Bojkova et al., 2020; Gordon et al., 2020).
The CoV membrane envelope has 3 primary structural proteins: spike (S), membrane (M), and envelope (E) (Boson et al., 2021; Siu et al., 2008; V’kovski et al., 2021) as well as accessory proteins such as Orf3a (Ito et al., 2005; Tan et al., 2004). Viral assembly only requires E and M (Boson et al., 2021; Siu et al., 2008; Vennema et al., 1996).
Yet, S is essential for infectivity as it mediates attachment to host cell receptors, angiotensin-converting enzyme 2 (ACE2) for SARS-CoV-2 (Hoffmann et al., 2020b; Letko et al., 2020), and fusion between viral and target cell membranes.
S, E, and M are synthesized in the ER, where folding and quaternary assembly occurs. They may also undergo post-translation modifications such as N-glycosylation on their luminal domains (Watanabe et al., 2020) and S-acylation (commonly referred to as S-palmitoylation) on their cytosolic parts, which occurs for spike and E from different CoVs (Boscarino et al., 2008; Lopez et al., 2008; McBride and Machamer, 2010; Petit et al., 2007; Thorp et al., 2006; Tseng et al., 2014).
S-acylation, which consists in the covalent attachment of medium-chain fatty acids (frequently palmitate, C16) to the sulfur atom of cytosolic cysteines, is very frequent in mammalian cells and a hallmark of viral envelope proteins (Gadalla and Veit, 2020; Schmidt, 1982).
S-acylation of spike was proposed to mediate its association to lipid microdomains, promote syncytia formation, and increase infectivity of mouse hepatitis virus (MHV) and pseudotyped particles (McBride and Machamer, 2010; Nguyen et al., 2020; Sanders et al., 2020; Thorp et al., 2006).
Here, we present a comprehensive study of spike S-acylation, the identification of the host enzymes involved, and its importance for viral biogenesis and infection. We identify ZDHHC20 as the dominant acyltransferase responsible for lipidation of spike, which plays several roles.
In the infected cell, it promotes spike biogenesis by protecting it from premature ER degradation, increases its half-life, and controls the lipid organization of its immediate membrane environment. Once the virus has formed, spike S-acylation controls fusion with the target cell.
Lipidomic analyses of SARS-CoV-2 viruses reveal a unique lipid composition, enriched in specific early Golgi sphingolipids. Finally, we show that S-acylation and sphingolipid metabolism promote SARS-CoV-2 infection, pointing toward S-acylation and lipid biogenesis related enzymes as novel potential anti-viral targets.
Here, based on a combination of functional genomics, biochemical, kinetic, and computational studies, we show that during SARS-CoV-2 biogenesis in infected cells, spike protein is rapidly and efficiently S-acylated following its synthesis in the ER, through the sequential action of the ZDHHC20 and ZDHHC9 enzymes.
Acylation prevents premature degradation promoting the biogenesis of spike, which is subsequently transported to the ERGIC where it arrives with up to 30 acyl chains decorating each trimer. The presence of these saturated lipid attracts cholesterol (Lenventhal et al., 2020), driving the formation of specific domains around spike. Using VLPs pseudotyped with WT or acylation-deficient spike, we could show that acylation is required for efficient viral fusion.
Our findings are consistent and complementary to those by Brangwynne and co-workers who recently identified that cholesterol present in the membrane of PPs is critical for spike-mediated fusion (Sanders et al., 2020). Our study shows that S-acylation constitutes a promising drug target for coronavirus infection.
ZDHHC20 is also involved in the acylation of the hemagglutinin of influenza virus (Gadalla et al., 2020), and the recent elucidation of its structure indicates that this enzyme should be specifically druggable (Rana et al., 2018). This study sets the ground for a re-emergent interest in the study of S-acylation and also lipid biosynthetic pathways as important regulatory mechanisms of infection by coronaviruses and enveloped viruses in general.