The SARS-CoV-2 spike glycoprotein interacts with MAO-B and impairs mitochondrial energetics.

SARS-CoV-2 infection is associated with both acute and post-acute neurological symptoms. Emerging evidence suggests that SARS-CoV-2 can alter mitochondrial metabolism, suggesting that changes in brain metabolism may contribute to the development of acute and post-acute neurological complications. Monoamine oxidase B (MAO-B) is a flavoenzyme located on the outer mitochondrial membrane that catalyzes the oxidative deamination of monoamine neurotransmitters. Computational analyses have revealed high similarity between the SARS-CoV-2 spike glycoprotein receptor binding domain on the ACE2 receptor and MAO-B, leading to the hypothesis that SARS-CoV-2 spike glycoprotein may alter neurotransmitter metabolism by interacting with MAO-B. Our results empirically establish that the SARS-CoV-2 spike glycoprotein interacts with MAO-B, leading to increased MAO-B activity in SH-SY5Y neuron-like cells. Common to neurodegenerative disease pathophysiological mechanisms, we also demonstrate that the spike glycoprotein impairs mitochondrial bioenergetics, induces oxidative stress, and perturbs the degradation of depolarized aberrant mitochondria through mitophagy. Our findings also demonstrate that SH-SY5Y neuron-like cells expressing the SARS-CoV-2 spike protein were more susceptible to MPTP-induced necrosis, likely necroptosis. Together, these results reveal novel mechanisms that may contribute to SARS-CoV-2-induced neurodegeneration.

Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells.

Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these 'late-penetrating viruses' must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Identification of a High-Frequency Intrahost SARS-CoV-2 Spike Variant with Enhanced Cytopathic and Fusogenic Effects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that is continuously evolving. Although its RNA-dependent RNA polymerase exhibits some exonuclease proofreading activity, viral sequence diversity can be produced by replication errors and host factors. A diversity of genetic variants can be observed in the intrahost viral population structure of infected individuals. Most mutations will follow a neutral molecular evolution and will not make significant contributions to variations within and between infected hosts. Herein, we profiled the intrasample genetic diversity of SARS-CoV-2 variants, also known as quasispecies, using high-throughput sequencing data sets from 15,289 infected individuals and infected cell lines. Despite high mutational background, we identified recurrent intragenetic variable positions in the samples analyzed, including several positions at the end of the gene encoding the viral spike (S) protein. Strikingly, we observed a high frequency of C→A missense mutations resulting in the S protein lacking the last 20 amino acids (SΔ20). We found that this truncated S protein undergoes increased processing and increased syncytium formation, presumably due to escaping M protein retention in intracellular compartments. Our findings suggest the emergence of a high-frequency viral sublineage that is not horizontally transmitted but potentially involved in intrahost disease cytopathic effects. IMPORTANCE The mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not make significant contributions to viral molecular evolution, some are naturally selected and produce variants through positive selection. Many SARS-CoV-2 variants have been recently described and show phenotypic selection toward more infectious viruses. Our study describes another type of variant that does not contribute to interhost heterogeneity but rather phenotypic selection toward variants that might have increased cytopathic effects. We identified that a C-terminal truncation of the spike protein removes an important endoplasmic reticulum (ER) retention signal, which consequently results in a spike variant that easily travels through the Golgi complex toward the plasma membrane in a preactivated conformation, leading to increased syncytium formation.

Ebola virus triggers receptor tyrosine kinase-dependent signaling to promote the delivery of viral particles to entry-conducive intracellular compartments.

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.

A Diacylglycerol Kinase Inhibitor, R-59-022, Blocks Filovirus Internalization in Host Cells.

Filoviruses, such as Ebola virus (EBOV) and Marburg virus, are causative agents of unpredictable outbreaks of severe hemorrhagic fevers in humans and non-human primates. For infection, filoviral particles need to be internalized and delivered to intracellular vesicles containing cathepsin proteases and the viral receptor Niemann-Pick C1. Previous studies have shown that EBOV triggers macropinocytosis of the viral particles in a glycoprotein (GP)-dependent manner, but the molecular events required for filovirus internalization remain mostly unknown. Here we report that the diacylglycerol kinase inhibitor, R-59-022, blocks EBOV GP-mediated entry into Vero cells and bone marrow-derived macrophages. Investigation of the mode of action of the inhibitor revealed that it blocked an early step in entry, more specifically, the internalization of the viral particles via macropinocytosis. Finally, R-59-022 blocked viral entry mediated by a panel of pathogenic filovirus GPs and inhibited growth of replicative Ebola virus. Taken together, our studies suggest that R-59-022 could be used as a tool to investigate macropinocytic uptake of filoviruses and could be a starting point for the development of pan-filoviral therapeutics.