The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

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.

Proprotein Convertases (PCs) are serine endoproteases that regulate the homeostasis of protein substrates in the cell. The PCs family counts 9 members-PC1/3, PC2, PC4, PACE4, PC5/6, PC7, Furin, SKI-1/S1P, and PCSK9. The first seven PCs are known as Basic Proprotein Convertases due to their propensity to cleave after polybasic clusters. SKI-1/S1P requires the additional presence of hydrophobic residues for processing, whereas PCSK9 is catalytically dead after autoactivation and exerts its functions using mechanisms alternative to direct cleavage. All PCs traffic through the canonical secretory pathway, reaching different compartments where the various substrates reside. Despite PCs members do not share the same subcellular localization, most of the cellular organelles count one or more Proprotein Convertases, including ER, Golgi stack, endosomes, secretory granules, and plasma membranes. The widespread expression of these enzymes at the systemic level speaks for their importance in the homeostasis of a large number of biological functions. Among others, PCs cleave precursors of hormones and growth factors and activate receptors and transcription factors. Notably, dysregulation of the enzymatic activity of Proprotein Convertases is associated to major human pathologies, such as cardiovascular diseases, cancer, diabetes, infections, inflammation, autoimmunity diseases, and Parkinson. In the current COVID-19 pandemic, Furin has further attracted the attention as a key player for conferring high pathogenicity to SARS-CoV-2. Here, we review the Proprotein Convertases family and their most important substrates along the secretory pathway. Knowledge about the complex functions of PCs is important to identify potential drug strategies targeting this class of enzymes.

Among the main metabolic pathways implicated in cancer cell proliferation are those of cholesterol and fatty acid synthesis, both of which are tightly regulated by sterol regulatory element-binding proteins (SREBPs). SREBPs are activated through specific cleavage by membrane-bound transcription factor protease 1 (MBTPS1), a serine protease that cleaves additional substrates (ATF6, BDNF, CREBs and somatostatin), some of which are also implicated in cell proliferation. The goal of this study was to determine whether MBTPS1 may serve as a master regulator in proliferation of colorectal cancer (CRC). Tumors from CRC patients showed variable levels of MBTPS1 mRNA, which were in positive correlation with the levels of SREBPs and ATF6, and in reverse correlation with BDNF levels. Chemical inhibition of MBTPS1 activity in two CRC-derived cell lines resulted in a marked decrease in the levels of SREBPs, but not of its other substrates and a marked decrease in cell proliferation, which suggested that MBTPS1 activity is critical for proliferation of these cells. In accordance, CRISPR/Cas9 targeted knockout (KO) of the MBTPS1 gene resulted in the survival of only a single clone that presented a phenotype of severely attenuated proliferation and marked downregulation of several energy metabolism pathways. We further showed that survival of the MBTPS1 KO clone was dependent upon significant upregulation of the type-1 interferon pathway, the inhibition of which halted proliferation entirely. Finally, rescue of the MBTPS1 KO cells, resulted in partial restoration of MBTPS1 levels, which was in accordance with partial recovery in proliferation and in SREBP levels. These finding suggest that MBTPS1 plays a critical role in regulating colon cancer proliferation primarily through SREBP-associated lipid metabolism, and as such may serve as a possible therapeutic target in CRC.

Inhibition of the binding of enveloped viruses surface glycoproteins to host cell receptor(s) is a major target of vaccines and constitutes an efficient strategy to block viral entry and infection of various host cells and tissues. Cellular entry usually requires the fusion of the viral envelope with host plasma membranes. Such entry mechanism is often preceded by \"priming\" and/or \"activation\" steps requiring limited proteolysis of the viral surface glycoprotein to expose a fusogenic domain for efficient membrane juxtapositions. The 9-membered family of Proprotein Convertases related to Subtilisin/Kexin (PCSK) serine proteases (PC1, PC2, Furin, PC4, PC5, PACE4, PC7, SKI-1/S1P, and PCSK9) participate in post-translational cleavages and/or regulation of multiple secretory proteins. The type-I membrane-bound Furin and SKI-1/S1P are the major convertases responsible for the processing of surface glycoproteins of enveloped viruses. Stefan Kunz has considerably contributed to define the role of SKI-1/S1P in the activation of arenaviruses causing hemorrhagic fever. Furin was recently implicated in the activation of the spike S-protein of SARS-CoV-2 and Furin-inhibitors are being tested as antivirals in COVID-19. Other members of the PCSK-family are also implicated in some viral infections, such as PCSK9 in Dengue. Herein, we summarize the various functions of the PCSKs and present arguments whereby their inhibition could represent a powerful arsenal to limit viral infections causing the present and future pandemics.

BACKGROUND: Arenaviruses are enveloped negative stranded viruses endemic in Africa, Europe and the Americas. Several arenaviruses cause severe viral hemorrhagic fever with high mortality in humans and pose serious public health threats. So far, there are no FDA-approved vaccines and therapeutic options are restricted to the off-label use of ribavirin. The major human pathogenic arenaviruses are classified as Category A agents and require biosafety level (BSL)-4 containment.
METHODS: Herein, the authors cover the recent progress in the development of BSL2 surrogate systems that recapitulate the entire or specific steps of the arenavirus life cycle and are serving as powerful platforms for drug discovery. Furthermore, they highlight the identification of selected novel drugs that target individual steps of arenavirus multiplication describing their discovery, their targets, and mode of action.
CONCLUSIONS: The lack of effective drugs against arenaviruses is an unmatched challenge in current medical virology. Novel technologies have provided important insights into the basic biology of arenaviruses and the mechanisms underlying virus-host cell interaction. Significant progress of our understanding of how the virus invades the host cell paved the way to develop powerful novel screening platforms. Recent efforts have provided a range of promising drug candidates currently under evaluation for therapeutic intervention in vivo.

Worldwide, approximately 170 million individuals are afflicted with chronic hepatitis C virus (HCV) infection. To prevent the development of inherent diseases such as cirrhosis and hepatocellular carcinoma, tremendous efforts have been made, leading to the development of promising new treatments. However, their efficiency is still dependent on the viral genotype. Additionally, these treatments that target the virus directly can trigger the emergence of resistant variants. In a previous study, we have demonstrated that a long-term (72h) inhibition of SKI-1/S1P, a master lipogenic pathway regulator through activation of SREBP, resulted in impaired HCV genome replication and infectious virion secretion. In the present study, we sought to investigate the antiviral effect of the SKI-1/S1P small molecule inhibitor PF-429242 at the early steps of the HCV lifecycle. Our results indicate a very potent antiviral effect of the inhibitor early in the viral lifecycle and that the overall action of the compound relies on two different contributions. The first one is SREBP/SKI-1/S1P dependent and involves LDLR and NPC1L1 proteins, while the second one is SREBP independent. Overall, our study confirms that SKI-1/S1P is a relevant target to impair HCV infection and that PF-429242 could be a promising candidate in the field of HCV infection treatment.