SKI-1/S1P Facilitates SARS-CoV-2 Spike Induced Cell-to-Cell Fusion via Activation of SREBP-2 and Metalloproteases, Whereas PCSK9 Enhances the Degradation of ACE2.

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2, which requires cleavage of its spike protein (S) at two sites: S1/S2 and S2'. This study investigates the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. The assays used were cell-to-cell fusion in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increased cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduced cell-to-cell fusion by promoting the cellular degradation of ACE2. SKI-1 activity led to enhanced S2' formation, which was attributed to increased metalloprotease activity as a response to enhanced cholesterol levels via activated SREBP-2. However, high metalloprotease activity resulted in the shedding of S2' into a new C-terminal fragment (S2″), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2″ formation abolished S2' and cell-to-cell fusion, as well as pseudoparticle entry, indicating that the formation of S2″ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested the binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms.

Proline-Proline Dyad in the Fusion Peptide of the Murine ß-Coronavirus Spike Protein's S2 Domain Modulates Its Neuroglial Tropism.

The ß-Coronavirus mouse hepatitis virus (MHV-A59)-RSA59 has a patent stretch of fusion peptide (FP) containing two consecutive central prolines (PP) in the S2 domain of the Spike protein. Our previous studies compared the PP-containing fusogenic-demyelinating strain RSA59(PP) to its one proline-deleted mutant strain RSA59(P) and one proline-containing non-fusogenic non-demyelinating parental strain RSMHV2(P) to its one proline inserted mutant strain RSMHV2(PP). These studies highlighted the crucial role of PP in fusogenicity, hepato-neuropathogenesis, and demyelination. Computational studies combined with biophysical data indicate that PP at the center of the FP provides local rigidity while imparting global fluctuation to the Spike protein that enhances the fusogenic properties of RSA59(PP) and RSMHV2(PP). To elaborate on the understanding of the role of PP in the FP of MHV, the differential neuroglial tropism of the PP and P mutant strains was investigated. Comparative studies demonstrated that PP significantly enhances the viral tropism for neurons, microglia, and oligodendrocytes. PP, however, is not essential for viral tropism for either astroglial or oligodendroglial precursors or the infection of meningeal fibroblasts in the blood-brain and blood-CSF barriers. PP in the fusion domain is critical for promoting gliopathy, making it a potential region for designing antivirals for neuro-COVID therapy.

SARS-CoV-2: A Master of Immune Evasion.

Viruses and their hosts have coevolved for a long time. This coevolution places both the pathogen and the human immune system under selective pressure; on the one hand, the immune system has evolved to combat viruses and virally infected cells, while viruses have developed sophisticated mechanisms to escape recognition and destruction by the immune system. SARS-CoV-2, the pathogen that is causing the current COVID-19 pandemic, has shown a remarkable ability to escape antibody neutralization, putting vaccine efficacy at risk. One of the virus's immune evasion strategies is mitochondrial sabotage: by causing reactive oxygen species (ROS) production, mitochondrial physiology is impaired, and the interferon antiviral response is suppressed. Seminal studies have identified an intra-cytoplasmatic pathway for viral infection, which occurs through the construction of tunneling nanotubes (TNTs), hence enhancing infection and avoiding immune surveillance. Another method of evading immune monitoring is the disruption of the antigen presentation. In this scenario, SARS-CoV-2 infection reduces MHC-I molecule expression: SARS-CoV-2's open reading frames (ORF 6 and ORF 8) produce viral proteins that specifically downregulate MHC-I molecules. All of these strategies are also exploited by other viruses to elude immune detection and should be studied in depth to improve the effectiveness of future antiviral treatments. Compared to the Wuhan strain or the Delta variant, Omicron has developed mutations that have impaired its ability to generate syncytia, thus reducing its pathogenicity. Conversely, other mutations have allowed it to escape antibody neutralization and preventing cellular immune recognition, making it the most contagious and evasive variant to date.

Two Consecutive Prolines in the Fusion Peptide of Murine ß-Coronavirus Spike Protein Predominantly Determine Fusogenicity and May Be Essential but Not Sufficient to Cause Demyelination.

Combined in silico, in vitro, and in vivo comparative studies between isogenic-recombinant Mouse-Hepatitis-Virus-RSA59 and its proline deletion mutant, revealed a remarkable contribution of centrally located two consecutive prolines (PP) from Spike protein fusion peptide (FP) in enhancing virus fusogenic and hepato-neuropathogenic potential. To deepen our understanding of the underlying factors, we extend our studies to a non-fusogenic parental virus strain RSMHV2 (P) with a single proline in the FP and its proline inserted mutant, RSMHV2 (PP). Comparative in vitro and in vivo studies between virus strains RSA59(PP), RSMHV2 (P), and RSMHV2 (PP) in the FP demonstrate that the insertion of one proline significantly resulted in enhancing the virus fusogenicity, spread, and consecutive neuropathogenesis. Computational studies suggest that the central PP in Spike FP induces a locally ordered, compact, and rigid structure of the Spike protein in RSMHV2 (PP) compared to RSMHV2 (P), but globally the Spike S2-domain is akin to the parental strain RSA59(PP), the latter being the most flexible showing two potential wells in the energy landscape as observed from the molecular dynamics studies. The critical location of two central prolines of the FP is essential for fusogenicity and pathogenesis making it a potential site for designing antiviral.

Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity.

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

Azadirachta indica A. Juss Ameliorates Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell Fusion in an Experimental Animal Model of Multiple Sclerosis.

Mouse hepatitis virus (MHV)-induced murine neuroinflammation serves as a model to study acute meningoencephalomyelitis, hepatitis, and chronic neuroinflammatory demyelination; which mimics certain pathologies of the human neurologic disease, multiple sclerosis (MS). MHV-induced acute neuroinflammation occurs due to direct glial cell dystrophy instigated by central nervous system (CNS)-resident microglia and astrocytes, in contrast to peripheral CD4+T cell-mediated myelin damage prevalent in the experimental autoimmune encephalomyelitis (EAE) model of MS. Viral envelope Spike glycoprotein-mediated cell-to-cell fusion is an essential mechanistic step for MHV-induced CNS pathogenicity. Although Azadirachta indica (Neem), a traditional phytomedicine, is known for its anti-inflammatory, anti-fungal, and spermicidal activities, not much is known about anti-neuroinflammatory properties of its bark (NBE) in MHV-induced acute neuroinflammation and chronic demyelination. Recombinant demyelinating MHV strain (RSA59) was preincubated with NBE to arrest the infection-initiation event, and its effect on viral replication, viral transcription, cytokine expression, and successive pathogenicity were investigated in vitro and in vivo. Virus-free Luciferase assay explained NBE's anti-virus-to-cell fusion activity in vitro. Intracranial inoculation of RSA59 preincubated with NBE into the mouse brain significantly reduces acute hepatitis, meningoencephalomyelitis, and chronic progressive demyelination. Additionally, NBE effectively restricts viral entry, dissemination in CNS, viral replication, viral transcription, and expression of the viral nucleocapsid and inflammatory cytokines. From mechanistic standpoints, RSA59 preincubated with NBE reduced viral entry, viral replication and cell-to-cell fusion, as a mode of viral dissemination. Moreover, intraperitoneal injection with NBE (25 mg/kg B.W.) into mice revealed a significant reduction in viral Nucleocapsid protein expression in vivo. Conclusively, A. indica bark extract may directly bind to the virus-host attachment Spike glycoprotein and suppresses MHV-induced neuroinflammation and neuropathogenesis by inhibiting cell-to-cell fusion and viral replication. Further studies will focus on combining bioanalytical assays to isolate potential NBE bioactive compound(s) that contribute towards the anti-viral activity of NBE.

Trypsin promotes porcine deltacoronavirus mediating cell-to-cell fusion in a cell type-dependent manner.

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.