ACE2-EGFR-MAPK signaling contributes to SARS-CoV-2 infection.

SARS-CoV-2 triggered the most severe pandemic of recent times. To enter into a host cell, SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2). However, subsequent studies indicated that other cell membrane receptors may act as virus-binding partners. Among these receptors, the epidermal growth factor receptor (EGFR) was hypothesized not only as a spike protein binder, but also to be activated in response to SARS-CoV-2. In our study, we aim at dissecting EGFR activation and its major downstream signaling pathway, the mitogen-activated signaling pathway (MAPK), in SARS-CoV-2 infection. Here, we demonstrate the activation of EGFR-MAPK signaling axis by the SARS-CoV-2 spike protein and we identify a yet unknown cross talk between ACE2 and EGFR that regulated ACE2 abundance and EGFR activation and subcellular localization, respectively. By inhibiting the EGFR-MAPK activation, we observe a reduced infection with either spike-pseudotyped particles or authentic SARS-CoV-2, thus indicating that EGFR serves as a cofactor and the activation of EGFR-MAPK contributes to SARS-CoV-2 infection.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Zika virus replication in glioblastoma cells: electron microscopic tomography shows 3D arrangement of endoplasmic reticulum, replication organelles, and viral ribonucleoproteins.

Structural changes of two patient-derived glioblastoma cell lines after Zika virus infection were investigated using scanning transmission electron tomography on high-pressure-frozen, freeze-substituted samples. In Zika-virus-infected cells, Golgi structures were barely visible under an electron microscope, and viral factories appeared. The cytosol outside of the viral factories resembled the cytosol of uninfected cells. The viral factories contained largely deranged endoplasmic reticulum (ER), filled with many so-called replication organelles consisting of a luminal vesicle surrounded by the ER membrane. Viral capsids were observed in the vicinity of the replication organelles (cell line #12537 GB) or in ER cisternae at large distance from the replication organelles (cell line #15747 GB). Near the replication organelles, we observed many about 100-nm-long filaments that may represent viral ribonucleoprotein complexes (RNPs), which consist of the RNA genome and N protein oligomers. In addition, we compared Zika-virus-infected cells with cells infected with a phlebovirus (sandfly fever Turkey virus). Zika virions are formed in the ER, whereas phlebovirus virions are assembled in the Golgi apparatus. Our findings will help to understand the replication cycle in the virus factories and the building of the replication organelles in glioblastoma cells.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

Increased in vitro Anti-HIV Activity of Caffeinium-Functionalized Polyoxometalates.

Polyoxometalates (POMs), molecular metal oxide anions, are inorganic clusters with promising antiviral activity. Herein we report increased anti-HIV-1 activity of a POM when electrostatically combined with organic counter-cations. To this end, Keggin-type cerium tungstate POMs have been combined with organic methyl-caffeinium (Caf) cations, and their cytotoxicity, antiviral activity and mode of action have been studied. The novel compound, Caf4 K[ß2 -CeSiW11 O39 ]×H2 O, exhibits sub-nanomolar antiviral activity and inhibits HIV-1 infectivity by acting on an early step of the viral infection cycle. This work demonstrates that combination of POM anions and organic bioactive cations can be a powerful new strategy to increase antiviral activity of these inorganic compounds.

Negatively Charged Peptide Nanofibrils from Immunoglobulin Light Chain Sequester Viral Particles but Lack Cell-Binding and Viral Transduction-Enhancing Properties.

Positively charged naturally occurring or engineered peptide nanofibrils (PNF) are effective enhancers of lentiviral and retroviral transduction, an often rate-limiting step in gene transfer and gene therapy approaches. These polycationic PNF are thought to bridge the electrostatic repulsions between negatively charged membranes of virions and cells, thereby enhancing virion attachment to and infection of target cells. Here, we analyzed PNF, which are formed by the peptide AL1, that represents a fragment of an immunoglobulin light chain that causes systemic AL amyloidosis. We found that negatively charged AL1 PNF interact with viral particles to a comparable extent as positively charged PNF. However, AL1 PNF lacked cell-binding activity, and consequently, did not enhance retroviral infection. These findings show that virion capture and cell binding of PNF are mediated by different mechanisms, offering avenues for the design of advanced PNF with selective functions.

Carrageenan-containing over-the-counter nasal and oral sprays inhibit SARS-CoV-2 infection of airway epithelial cultures.

Pharmaceutical interventions are urgently needed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission. As SARS-CoV-2 infects and spreads via the nasopharyngeal airways, we analyzed the antiviral effect of selected nasal and oral sprays on virus infection in vitro. Two nose sprays showed virucidal activity but were cytotoxic precluding further analysis in cell culture. One nasal and one mouth spray suppressed SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells and primary differentiated human airway epithelial cultures. The antiviral activity in both sprays could be attributed to polyanionic ι- and κ-carrageenans. Thus, application of carrageenan-containing nasal and mouth sprays may reduce the risk of acquiring SARS-CoV-2 infection and may limit viral spread, warranting further clinical evaluation.

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

Drug Inhibition of SARS-CoV-2 Replication in Human Pluripotent Stem Cell-Derived Intestinal Organoids.

BACKGROUND AND AIMS: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need. METHODS: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. RESULTS: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. CONCLUSIONS: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Guanylate-Binding Proteins 2 and 5 Exert Broad Antiviral Activity by Inhibiting Furin-Mediated Processing of Viral Envelope Proteins.

Guanylate-binding protein (GBP) 5 is an interferon (IFN)-inducible cellular factor reducing HIV-1 infectivity by an incompletely understood mechanism. Here, we show that this activity is shared by GBP2, but not by other members of the human GBP family. GBP2/5 decrease the activity of the cellular proprotein convertase furin, which mediates conversion of the HIV-1 envelope protein (Env) precursor gp160 into mature gp120 and gp41. Because this process primes HIV-1 Env for membrane fusion, viral particles produced in the presence of GBP2/5 are poorly infectious due to increased incorporation of non-functional gp160. Furin activity is critical for the processing of envelope glycoproteins of many viral pathogens. Consistently, GBP2/5 also inhibit Zika, measles, and influenza A virus replication and decrease infectivity of viral particles carrying glycoproteins of Marburg and murine leukemia viruses. Collectively, our results show that GPB2/5 exert broad antiviral activity by suppressing the activity of the virus-dependency factor furin.

Natural Inhibitor of Human Cytomegalovirus in Human Seminal Plasma.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital infections that can lead to severe birth defects. Although HCMV is frequently detected in semen and thus is potentially sexually transmitted, the role of semen in HCMV transmission is largely unclear. Here we describe that human seminal plasma (SP; the cell-free supernatant of semen) inhibits HCMV infection. The inhibition of HCMV infection was dose dependent and effective for different cell types, virus strains, and semen donors. This inhibitory effect was specific for HCMV, as herpes simplex virus 2 (HSV-2) and human immunodeficiency virus type 1 (HIV-1) infections were enhanced by SP. Mechanistically, SP inhibited infection by interfering with the attachment of virions to cells most likely via an interaction with the trimeric glycoprotein complex gH/gL/gO. Together, our findings suggest that semen contains a factor that potentially limits sexual transmission of HCMV.IMPORTANCE The role of semen in sexual transmission of human cytomegalovirus (HCMV) is currently unclear. This is surprising, as HCMV is frequently detected in this body fluid and infection is of high danger for neonates and pregnant women. In this study, we found that seminal plasma (SP) dose dependently inhibited HCMV infection. The infection inhibition was specific for HCMV, as other viruses, such as human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus 2 (HSV-2), were not inhibited by SP. SP must contain a soluble, heat-resistant factor that limits attachment of HCMV particles to cells, probably by interaction with the trimeric glycoprotein complex gH/gL/gO. This novel virus-host interaction could possibly limit transmission of HCMV via semen during sexual intercourse.

Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure.

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

Semen inhibits Zika virus infection of cells and tissues from the anogenital region.

Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.

Altered cytokine responses to cognitive stress in multiple sclerosis patients with fatigue.

This study intended to examine if the immune response to a cognitive task as a variant of psychological stress in MS patients is distinct from healthy controls. The experiment was part of a larger study on mechanisms and measurements of MS fatigue. Patients (n =23) and controls (n =25) participated in a cognitive task lasting 40 minutes, in which the heart rate was continuously monitored. Blood samples were taken at baseline and directly after the stress-inducing task Whole blood stimulated cytokine production representative of the TH-1 (i.e. IFNgamma, TNFalpha) and TH-2 paradigm (i.e. IL-10) was evaluated in relation to disability, fatigue, cognitive deficit, and anxiety. Patients scored high on a disease specific fatigue score compared to controls, whereas baseline cytokine patterns did not differ between the groups. MS patients displayed a blunted response of IFNgamma (P =0.03) whereas TNFalpha and IL-10 responses did not change. Additionally MS patients showed a significantly lower heart rate increase after the task (P <0.001). Cognitive impairment was associated with a decreased heart rate reactivity (P =0.02) while depressive symptoms correlated with stronger IL-10 responses (P =0.05). Overall, cognitive stress induces IFNgamma production in healthy controls but not in MS patients with fatigue. Furthermore, a reduced cardiac response might indicate an autonomic dysfunction in this group of patients.