Abstract
One of the outstanding mysteries of COVID-19 is how the SARS-CoV-2 virus can cause so much damage throughout the body—heart, liver, brain, kidneys, and the list goes on—without necessarily infecting all of those tissues. A recent study by Abhijit Basu et al. has given us new insight into how, at least some of this damage, might occur. The key discovery is that the SARS-CoV-2 virus need not enter a cell to disturb its function in profound ways; it may actually be sufficient for the spike (S) protein to bind to the outside of a cell to cause significant, and even lethal, changes. Although the puzzle is far from complete, their work shines some light into a dark corner of COVID-19 pathogenesis.
Background: Autoregulation of inflammation
This story begins almost two decades ago. Barsanjit Mazumder, senior author of this new study, and his colleagues at the time were interested in how our body autoregulates inflammation—not only how it's initiated but, importantly, how it's controlled and how it's turned off once the threat has been cleared. What turns on inflammation is well known. Much about what turns it off remains to be discovered.
Interferon-gamma (IFN-γ) acts both to stimulate and inhibit inflammation, in part by inducing and then silencing the inflammatory protein ceruloplasmin. Ceruloplasmin is produced by macrophages following IFN-γ activation and contributes to the host immune response. As with other inflammatory proteins, ceruloplasmin creates a toxic microenvironment. The flip side of this defensive strategy? It can also end up damaging our own bodies if left unchecked.
Mazumder and his fellow researchers analyzed the potential for interferon-gamma to downregulate ceruloplasmin. They made a startling discovery: IFN-γ-induced extracellular signaling can silence ceruloplasmin mRNA translation, halting its production.
Fourteen to fifteen hours after initial stimulation, IFN-γ activates death-associated protein kinase-1 (DAPK-1). DAPK-1 is a key player in the regulation of apoptosis, a process by which stressed cells commit suicide in such a way as to not induce inflammation; they just quietly digest themselves. DAPK-1, in turn, activates another kinase, zipper-interacting protein kinase (ZIPK), which then phosphorylates a 60S-associated ribosomal protein, L13a.
Once phosphorylated, L13a leaves the ribosome to aggregate with three other proteins—glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—to form a complex, the interferon-gamma activated inhibitor of translation (GAIT) complex, or simply GAIT.
A key discovery is that GAIT silences ceruloplasmin synthesis (Figure 1) by binding to a 3 prime stem loop of the mRNA (Figure 2). They named this hairpin RNA structure the GAIT element. We now know that many gamma-interferons are down-regulated by similar GAIT-like elements.
A representation of the signaling pathway discovered by Mazumder and his colleagues, showing: a) induction and translation of ceruloplasmin mRNA, b) phosphorylation of L13a, and c) silencing of ceruloplasmin mRNA translation by the GAIT protein complex. FROM: “Regulated Release of L13a from the 60S Ribosomal Subunit as A Mechanism of Transcript-Specific Translational Control” MAZUMDER ET AL. 2003
Schematic of the GAIT hairpin loop structure of human ceruloplasmin mRNA. FROM: “A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells” ABHIJIT BASU ET AL. 2022
A GAIT-like element inhibits messenger RNA expression of a pig coronavirus
Several years later, Fernando Almazán and his group in Spain noticed that a pig coronavirus, transmissible gastroenteritis coronavirus (TGEV), has a 32-nucleotide RNA motif at the 3′-UTR end of its genome that very closely resembles the GAIT element found in ceruloplasmin, both in structure as well as in sequence (Figure 3).
(Top) TGEV genome, with a sequence comparison between the F2.2L fragment and that of the canonical GAIT element of human ceruloplasmin. (Bottom) A comparison of the hairpin loop structures of the GAIT-like element in TGEV and canonical ceruloplasmin GAIT element. FROM: “Identification of a Gamma Interferon-Activated Inhibitor of Translation-Like RNA Motif at the 3′ End of the Transmissible Gastroenteritis Coronavirus Genome Modulating Innate Immune Response” ALMAZÁN ET AL. 2015
The TGEV GAIT-like element specifically interacts with two host proteins: glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). In vitro tests revealed that these proteins directly bind the RNA motif, silencing translation of those mRNAs containing the motif. Almazán et al. engineered a mutant version of the TGEV virus that lacked the GAIT-like RNA motif. The mutant virus elicited a much stronger immune response than did the parent. The authors concluded that the GAIT-like elements act to suppress the host's immune response.
Spike-ACE2 interactions in SARS-CoV-1
The final hint came from a group of researchers in Taiwan. The cellular protein ACE2 serves as the receptor for both SARS-CoV-1 and -2. Chen et al. noticed that, in some circumstances, binding of ACE2 by the SARS-CoV-1 spike also triggers a kinase cascade altering cellular and viral functions. They found that S protein binding to ACE 2 upregulates the expression of the chemokine ligand 2 (CCL2), a small protein that usually recruits immune cells—monocytes, dendritic cells, and memory T cells—to sites of inflammation. CCL2 is also associated with lung inflammatory disorders including asthma, respiratory distress syndrome, and pulmonary fibrosis. CCL2 knockout mice show a significant reduction in lung fibrosis when compared to their wild-type counterparts.
Chen et al. worked backwards from CCL2 activation to elucidate the signal transduction pathway from S protein binding to gene activation. They determined that activator protein 1 (AP-1) activates the CCL2 promoter upon SARS-CoV-1 infection. AP-1, in turn, is regulated by a set of proteins called mitogen-activated protein kinases (MAPKs). Two of these turned out to be particularly relevant, ERK1 and ERK2. Inhibition of either completely halts CCL2 upregulation.
The full pathway by which S protein ACE2 binding triggers CCL2 production is shown in figure 4. Key players include: casein kinase 2 (CK2) phosphorylation of the cytoplasmic tail of ACE2, the Ras-Raf-MEK-ERK signaling cascade, and AP-1 activation of CCL2 mRNA. Chen et al. suggest that the upregulation of CCL2 by SARS-CoV-1 contributes to SARS lung fibrosis.
Schematic of the Ras-ERK-AP-1 pathway through which SARS-CoV-1 triggers CCL2 upregulation in human lung epithelial cells. FROM: “Upregulation of the Chemokine (C-C Motif) Ligand 2 via a Severe Acute Respiratory Syndrome Coronavirus Spike-ACE2 Signaling Pathway” I-YIN CHEN ET AL. 2010
S protein inhibition of SARS-CoV-2 messenger RNA expression
Basu et al. noticed that the genome of SARS-CoV-2 has two sequences, one located within ORF1a and a second in the S gene, that could form hairpin loop structures akin to those of canonical GAIT elements (Figures 5 & 6), albeit with dissimilar genetic sequences.
Schematic representation showing the location of ORF1a RNA and Spike mRNA within the SARS-CoV-2 genome. BASU ET AL. 2022
GAIT-like hairpin loop structures in SARS-CoV-2 ORF1a RNA and S mRNA. BASU ET AL. 2022
To test the possibility that these sequences also respond to SARSCoV-2's spike binding to ACE2, Basu et al. inserted these GAIT-like elements into the 3' UTR of luciferase mRNA in cell lines that express ACE2. They report that S protein binding to ACE2 silences both luciferase constructs. They also find that S protein presented either on a virus-like particle or on the surface of a lentivirus vector silences constructs carrying either of the two GAIT-like elements. To distinguish these SARS-CoV-2 sequences from their cellular counterparts, they named them “virus-activated inhibitor of translation (VAIT)” elements.
Like GAIT, both SARS-CoV-2 VAIT elements are bound by a multi-protein complex including the phosphorylated L13A protein. Curiously, despite similar secondary stem loop structures, none of the three elements, the ceruloplasmin GAIT element, nor the two SARS-CoV-2 VAIT elements, compete with one another for binding by the L13a complex.
Basu et al. speculate that both the pathway that silences ceruloplasmin and the one that upregulates CCL2 during SARS-CoV-1 infection contribute to the silencing of VAIT element-containing SARS-CoV-2 messenger RNAs. The VAIT pathway is similar to the one that silences ceruloplasmin insofar as they both rely on DAPK-1 kinase to phosphorylate the ribosomal protein L13a. It is similar to the SARS-CoV-1 pathway in its dependence on CK2 and ERK1/2 to kickstart signaling after initial S-ACE2 interaction. Formation of the phosphorylated L13a complex is inhibited either by a drug that blocks DAPK-1 or by DAPK-1 depletion. Figure 7 outlines a proposed pathway for VAIT element binding.
A model of the VAIT-dependent signaling pathway that silences synthesis of SARS-CoV-2 Orfa1 and S proteins in host cells (human lung epithelial cells). BASU ET AL. 2022
Conclusion
These studies show that, independent of its role as an entry receptor for SARS-CoV-1 and -2, extracellular binding of the spike protein to host ACE2 triggers an intracellular cascade that has the potential to alter both cell physiology and expression of viral proteins. The SARS-CoV-1 S protein triggers expression of an active cytokine, CCL2, and S-ACE2 interactions in SARS-CoV-2 may downregulate expression of Orf and S proteins.
An independent study by Alovio et al. found that SARS-CoV-2 spike binding to another receptor, C147, initiates a signaling cascade that disrupts pericyte function and induces death of vascular endothelial cells. Together these studies show that signal transduction induced by sarbecovirus S protein binding to surface receptors are likely to contribute to viral pathogenesis independent of the virus's ability to enter and to replicate in the target cell.