Factors associated with health professionals' stress reactions, job satisfaction, intention to leave and health-related outcomes in acute care, rehabilitation and psychiatric hospitals, nursing homes and home care organisations.

The aim of this study is to identify (1) the extent of work-related stress and (2) stressors associated with cognitive and behavioral stress reactions, burnout symptoms, health status, quality of sleep, job satisfaction, and intention to leave the organization and the profession among health professionals working in acute care /rehabilitation hospitals, psychiatric hospitals, nursing homes, and home care organizations. BACKGROUND: Health professionals are faced with various stressors at work and as a consequence are leaving their profession prematurely. This study aimed to identify the extent of work-related stress and stressors associated with stress reactions, job satisfaction, and intention to leave and health-related outcomes among health professionals working in different healthcare sectors (acute care, rehabilitation and psychiatric hospitals, nursing homes and home care organizations). METHODS: This study is based on a repeated cross-sectional design, which includes three data measures between 2017 and 2020 and 19,340 participating health professionals from 26 acute care / rehabilitation hospitals, 12 psychiatric hospitals, 86 nursing homes and 41 home care organizations in Switzerland. For data analysis, hierarchical multilevel models (using AIC) were calculated separately for hospitals, nursing homes, and home care organizations, regarding health professionals' stress symptoms, job satisfaction, intention to leave the organization / profession, general health status, burnout symptoms, and quality of sleep. RESULTS: The main findings reveal that the incompatibility of health professionals' work and private life was significantly associated (p < 0.05) with their stress reactions, job satisfaction, intention to leave, and health-related outcomes in all the included work areas. The direct supervisor's good leadership qualities were also associated with health professionals' job satisfaction regarding all work areas (B ≥ 0.22, p = 0.000). In addition, a positive perceived bond with the organization (B ≥ 0.13, p < 0.01) and better development opportunities (B ≥ 0.05, p < 0.05) were associated with higher job satisfaction and a lower intention to leave the organization and profession among health professionals. Also, a younger age of health professionals was associated with a higher intention to leave the organization and the profession prematurely in all the included work areas. High physical (B ≥ 0.04, p < 0.05) and quantitative demands (B ≥ 0.05, p = 0.000) at work were also associated with negative health-related outcomes.

Setting new priorities for nursing research: The updated Swiss Nursing Research Agenda-a systematic, participative approach.

AIM: To identify current key areas for nursing research in Switzerland, we revised the Swiss Research Agenda for Nursing (SRAN) initially published in 2008. BACKGROUND: By developing a research agenda, nursing researchers internationally prioritize and cluster relevant topics within the research community. The process should be collaborative and systematic to provide credible information for decisionmakers in health care research, policy, and practice. SOURCES OF EVIDENCE: After a participative, systematic, and critical evaluation within and outside of the Swiss Association for Nursing Science, the updated SRAN 2019-2029 defines four research priorities (new models of care, nursing care interventions, work and care environment, and quality of care and patient safety) and four transversal themes (organization of research, research methodologies, research in health care policy and public health perspectives). CONCLUSION: Adding to other national nursing research agendas, the categories are organized in a framework of key research priorities and transversal themes. They relate to the importance of global and local foci of research as well as challenges in health care services and policy systems. The agenda is an important prerequisite for enhancing the influence of nursing research in Switzerland and provides guidance for the next decade. IMPLICATIONS FOR NURSING PRACTICE: The revised agenda ensures that research projects target key knowledge gaps and the discipline's core questions in respective countries. IMPLICATIONS FOR HEALTH POLICY: Nursing research should inform and influence health policy on all institutional and political levels. Therefore, the integration of public health perspectives in research is one of the most important new aspects of SRAN 2019-2029.

Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

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.

Comparison of the Innate Immune Responses to Pathogenic and Nonpathogenic Clade B New World Arenaviruses.

The New World (NW) arenaviruses are a diverse group of zoonotic viruses, including several causative agents of severe hemorrhagic fevers in humans. All known human-pathogenic NW arenaviruses belong to clade B, where they group into sublineages with phylogenetically closely related nonpathogenic viruses, e.g., the highly pathogenic Junin (JUNV) and Machupo viruses with the nonpathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of nonpathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cell's innate antiviral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune responses against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV infection did. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in the presence or absence of an IFN-I response. However, in comparison to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2α. Confocal microscopy studies revealed similar subcellular colocalizations of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV but promoted TCRV multiplication, providing the first evidence for differential innate recognition and control of pathogenic and nonpathogenic NW arenaviruses by PKR.IMPORTANCE New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of nonpathogenic NW arenaviruses with their highly pathogenic cousins suggests that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune responses against the highly pathogenic Junin virus (JUNV) and the related nonpathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivities to IFN-I treatment. However, the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) contributed to the control of TCRV, but not JUNV, providing the first evidence for differential innate recognition and control of JUNV and TCRV.

Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion.

Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses.IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.

Axl Can Serve as Entry Factor for Lassa Virus Depending on the Functional Glycosylation of Dystroglycan.

Fatal infection with the highly pathogenic Lassa virus (LASV) is characterized by extensive viral dissemination, indicating broad tissue tropism. The major cellular receptor for LASV is the highly conserved extracellular matrix receptor dystroglycan (DG). Binding of LASV depends on DG's tissue-specific posttranslational modification with the unusual O-linked polysaccharide matriglycan. Interestingly, functional glycosylation of DG does not always correlate with viral tropism observed in vivo The broadly expressed phosphatidylserine (PS) receptors Axl and Tyro3 were recently identified as alternative LASV receptor candidates. However, their role in LASV entry is not entirely understood. Here, we examine LASV receptor candidates in primary human cells and found coexpression of Axl with differentially glycosylated DG. To study LASV receptor use in the context of productive arenavirus infection, we employed recombinant lymphocytic choriomeningitis virus expressing LASV glycoprotein (rLCMV-LASV GP) as a validated biosafety level 2 (BSL2) model. We confirm and extend previous work showing that Axl can contribute to LASV entry in the absence of functional DG using "apoptotic mimicry" in a way similar to that of other enveloped viruses. We further show that Axl-dependent LASV entry requires receptor activation and involves a pathway resembling macropinocytosis. Axl-mediated LASV entry is facilitated by heparan sulfate and critically depends on the late endosomal protein LAMP-1 as an intracellular entry factor. In endothelial cells expressing low levels of functional DG, both receptors are engaged by the virus and can contribute to productive entry. In sum, we characterize the role of Axl in LASV entry and provide a rationale for targeting Axl in antiviral therapy.IMPORTANCE The highly pathogenic arenavirus Lassa virus (LASV) represents a serious public health problem in Africa. Although the principal LASV receptor, dystroglycan (DG), is ubiquitously expressed, virus binding critically depends on DG's posttranslational modification, which does not always correlate with tissue tropism. The broadly expressed phosphatidylserine receptor Axl was recently identified as an alternative LASV receptor candidate, but its role in LASV entry is unclear. Here, we investigate the exact role of Axl in LASV entry as a function of DG's posttranslational modification. We found that in the absence of functional DG, Axl can mediate LASV entry via apoptotic mimicry. Productive entry requires virus-induced receptor activation, involves macropinocytosis, and critically depends on LAMP-1. In endothelial cells that express low levels of glycosylated DG, both receptors can promote LASV entry. In sum, our study defines the roles of Axl in LASV entry and provides a rationale for targeting Axl in antiviral therapy.

Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction.

Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P).

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process.

Lassa Virus Cell Entry via Dystroglycan Involves an Unusual Pathway of Macropinocytosis.

UNLABELLED: The pathogenic Old World arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with a high rate of mortality in humans. Several LASV receptors, including dystroglycan (DG), TAM receptor tyrosine kinases, and C-type lectins, have been identified, suggesting complex receptor use. Upon receptor binding, LASV enters the host cell via an unknown clathrin- and dynamin-independent pathway that delivers the virus to late endosomes, where fusion occurs. Here we investigated the mechanisms underlying LASV endocytosis in human cells in the context of productive arenavirus infection, using recombinant lymphocytic choriomeningitis virus (rLCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that rLCMV-LASVGP entered human epithelial cells via DG using a macropinocytosis-related pathway independently of alternative receptors. Dystroglycan-mediated entry of rLCMV-LASVGP required sodium hydrogen exchangers, actin, and the GTPase Cdc42 and its downstream targets, p21-activating kinase-1 (PAK1) and Wiskott-Aldrich syndrome protein (N-Wasp). Unlike other viruses that enter cells via macropinocytosis, rLCMV-LASVGP entry did not induce overt changes in cellular morphology and hardly affected actin dynamics or fluid uptake. Screening of kinase inhibitors identified protein kinase C, phosphoinositide 3-kinase, and the receptor tyrosine kinase human hepatocyte growth factor receptor (HGFR) to be regulators of rLCMV-LASVGP entry. The HGFR inhibitor EMD 1214063, a candidate anticancer drug, showed antiviral activity against rLCMV-LASVGP at the level of entry. When combined with ribavirin, which is currently used to treat human arenavirus infection, EMD 1214063 showed additive antiviral effects. In sum, our study reveals that DG can link LASV to an unusual pathway of macropinocytosis that causes only minimal perturbation of the host cell and identifies cellular kinases to be possible novel targets for therapeutic intervention. IMPORTANCE: Lassa virus (LASV) causes several hundred thousand infections per year in Western Africa, with the mortality rate among hospitalized patients being high. The current lack of a vaccine and the limited therapeutic options at hand make the development of new drugs against LASV a high priority. In the present study, we uncover that LASV entry into human cells via its major receptor, dystroglycan, involves an unusual pathway of macropinocytosis and define a set of cellular factors implicated in the regulation of LASV entry. A screen of kinase inhibitors revealed HGFR to be a possible candidate target for antiviral drugs against LASV. An HGFR candidate inhibitor currently being evaluated for cancer treatment showed potent antiviral activity and additive drug effects with ribavirin, which is used in the clinic to treat human LASV infection. In sum, our study reveals novel fundamental aspects of the LASV-host cell interaction and highlights a possible candidate drug target for therapeutic intervention.

A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

UNLABELLED: Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. IMPORTANCE: Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human cells is the processing of the viral envelope glycoprotein by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). In order to break the species barrier during zoonotic transmission and cause severe disease in humans, newly emerging arenaviruses must be able to hijack human SKI-1/S1P efficiently. Here we implement a newly developed cell-based molecular sensor for human SKI-1/S1P to characterize the processing of arenavirus glycoproteins in a quantitative manner. We further use our sensor to correctly predict efficient processing of the glycoprotein of the newly emergent pathogenic Lujo virus by human SKI-1/S1P. Our sensor thus represents a rapid and robust test system with which to assess whether the glycoprotein of any newly emerging arenavirus can be efficiently processed by human SKI-1/S1P, based solely on sequence information.

Lymphocytic Choriomeningitis Virus Differentially Affects the Virus-Induced Type I Interferon Response and Mitochondrial Apoptosis Mediated by RIG-I/MAVS.

UNLABELLED: Arenaviruses are important emerging human pathogens maintained by noncytolytic persistent infection in their rodent reservoir hosts. Despite high levels of viral replication, persistently infected carrier hosts show only mildly elevated levels of type I interferon (IFN-I). Accordingly, the arenavirus nucleoprotein (NP) has been identified as a potent IFN-I antagonist capable of blocking activation of interferon regulatory factor 3 (IRF3) via the retinoic acid inducible gene (RIG)-I/mitochondrial antiviral signaling (MAVS) pathway. Another important mechanism of host innate antiviral defense is represented by virus-induced mitochondrial apoptosis via RIG-I/MAVS and IRF3. In the present study, we investigated the ability of the prototypic Old World arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with RIG-I/MAVS-dependent apoptosis. We found that LCMV does not induce apoptosis at any time during infection. While LCMV efficiently blocked induction of IFN-I via RIG-I/MAVS in response to superinfection with cytopathic RNA viruses, virus-induced mitochondrial apoptosis remained fully active in LCMV-infected cells. Notably, in LCMV-infected cells, RIG-I was dispensable for virus-induced apoptosis via MAVS. Our study reveals that LCMV infection efficiently suppresses induction of IFN-I but does not interfere with the cell's ability to undergo virus-induced mitochondrial apoptosis as a strategy of innate antiviral defense. The RIG-I independence of mitochondrial apoptosis in LCMV-infected cells provides the first evidence that arenaviruses can reshape apoptotic signaling according to their needs. IMPORTANCE: Arenaviruses are important emerging human pathogens that are maintained in their rodent hosts by persistent infection. Persistent virus is able to subvert the cellular interferon response, a powerful branch of the innate antiviral defense. Here, we investigated the ability of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with the induction of programmed cell death, or apoptosis, in response to superinfection with cytopathic RNA viruses. Upon viral challenge, persistent LCMV efficiently blocked induction of interferons, whereas virus-induced apoptosis remained fully active in LCMV-infected cells. Our studies reveal that the persistent virus is able to reshape innate apoptotic signaling in order to prevent interferon production while maintaining programmed cell death as a strategy for innate defense. The differential effect of persistent virus on the interferon response versus its effect on apoptosis appears as a subtle strategy to guarantee sufficiently high viral loads for efficient transmission while maintaining apoptosis as a mechanism of defense.

Integrated Control of Apple Scab and Powdery Mildew in an Organic Apple Orchard by Combining Potassium Carbonates with Wettable Sulfur, Pruning, and Cultivar Susceptibility.

In a 4-year study in a whole-field sanitized organic apple orchard, the effectiveness of nine fungicide treatments, including potassium mono- and bicarbonate and their combinations with wettable sulfur, were evaluated for scab and powdery mildew control on two cultivars with different susceptibility to scab and powdery mildew, under two pruning treatments. The whole-field sanitation practice was performed by removal of infected fallen leaves. Treatment effects on phytotoxicity and yield were also determined. Pruning significantly reduced leaf scab incidence but only on the more scab-susceptible Idared. Pruning significantly reduced mildew incidence in most years and on both cultivars but the more mildew-susceptible Jonathan showed significantly higher mildew incidence than Idared. Among products approved for organic production, the best scab control was achieved with a potassium mono- or bicarbonate treatment combined with wettable sulfur, except for Jonathan in 2011 on leaf and in 2014 on both leaf and fruit, and for Idared in 2013 on fruit. The best mildew control was also achieved with potassium mono- or bicarbonate treatments combined with wettable sulfur, with exceptions on shoots of Idared in 2011 and 2013 and on fruit of Idared in 2012. Leaf phytotoxicity was significantly higher in all potassium carbonate treatments compared with untreated plots, except for Idared in 2012, while fruit russet in these treatments did not differ significantly from the untreated plots. However, phytotoxicity values of all carbonate treatments were significantly lower than the lime sulfur treatment in most years on both cultivars. Yield of the potassium mono- or bicarbonate treatments combined with wettable sulfur was significantly higher than the untreated plots in the pruned treatments for both cultivars in all years. The integrated control approach designed for organic disease management against the two pathogens is discussed.

Zymogen activation and subcellular activity of subtilisin kexin isozyme 1/site 1 protease.

The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B'/B followed by the herein newly identified C'/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B'/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1-P8) and P1' are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates.

A dystroglycan mutation associated with limb-girdle muscular dystrophy.

Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192→Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.

Role of DC-SIGN in Lassa virus entry into human dendritic cells.

The arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DCs), are early and preferred targets of LASV, and their productive infection contributes to the virus-induced immunosuppression observed in fatal disease. Here, we characterized the role of the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in LASV entry into primary human DCs using a chimera of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that differentiation of human primary monocytes into DCs enhanced virus attachment and entry, concomitant with the upregulation of DC-SIGN. LASV and rLCMV-LASVGP bound to DC-SIGN via mannose sugars located on the N-terminal GP1 subunit of LASVGP. We provide evidence that DC-SIGN serves as an attachment factor for rLCMV-LASVGP in monocyte-derived immature dendritic cells (MDDC) and can accelerate the capture of free virus. However, in contrast to the phlebovirus Uukuniemi virus (UUKV), which uses DC-SIGN as an authentic entry receptor, productive infection with rLCMV-LASVGP was less dependent on DC-SIGN. In contrast to the DC-SIGN-mediated cell entry of UUKV, entry of rLCMV-LASVGP in MDDC was remarkably slow and depended on actin, indicating the use of different endocytotic pathways. In sum, our data reveal that DC-SIGN can facilitate cell entry of LASV in human MDDC but that its role seems distinct from the function as an authentic entry receptor reported for phleboviruses.

Differential recognition of Old World and New World arenavirus envelope glycoproteins by subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P).

The arenaviruses are an important family of emerging viruses that includes several causative agents of severe hemorrhagic fevers in humans that represent serious public health problems. A crucial step of the arenavirus life cycle is maturation of the envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). Comparison of the currently known sequences of arenavirus GPCs revealed the presence of a highly conserved aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in Old World and clade C New World arenaviruses but not in New World viruses of clades A and B or cellular substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function analysis, we found that residue Y285 of SKI-1/S1P, distal from the catalytic triad, is implicated in the molecular recognition of the aromatic "signature residue" at P7 in the GPC of Old World Lassa virus. Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1P is crucial for the efficient processing of peptides derived from Old World and clade C New World arenavirus GPCs but not of those from clade A and B New World arenavirus GPCs. The data suggest that during coevolution with their mammalian hosts, GPCs of Old World and clade C New World viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four-amino-acid recognition sequences and currently occupy an extended binding pocket.

Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection.

α-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of α-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on α-dystroglycan. Here we show that the LARGE modification required for laminin- and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of α-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of α-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr(317)ProThr(319)ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature α-dystroglycan and is crucial for α-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.

Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever.

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.

Biological and structural characterization of Trypanosoma cruzi phosphodiesterase C and Implications for design of parasite selective inhibitors.

Trypanosoma cruzi phosphodiesterase C (TcrPDEC) is a potential new drug target for the treatment of Chagas disease but has not been well studied. This study reports the enzymatic properties of various kinetoplastid PDECs and the crystal structures of the unliganded TcrPDEC1 catalytic domain and its complex with an inhibitor. Mutations of PDEC during the course of evolution led to inactivation of PDEC in Trypanosoma brucei/Trypanosoma evansi/Trypanosoma congolense, whereas the enzyme is active in all other kinetoplastids. The TcrPDEC1 catalytic domain hydrolyzes both cAMP and cGMP with a K(m) of 23.8 µm and a k(cat) of 31 s(-1) for cAMP and a K(m) of 99.1 µm and a k(cat) of 17 s(-1) for cGMP, thus confirming its dual specificity. The crystal structures show that the N-terminal fragment wraps around the TcrPDEC catalytic domain and may thus regulate its enzymatic activity via direct interactions with the active site residues. A PDE5 selective inhibitor that has an IC(50) of 230 nm for TcrPDEC1 binds to TcrPDEC1 in an orientation opposite to that of sildenafil. This observation, together with the screen of the inhibitory potency of human PDE inhibitors against TcrPDEC, implies that the scaffold of some human PDE inhibitors might be used as the starting model for design of parasite PDE inhibitors. The structural study also identified a unique parasite pocket that neighbors the active site and may thus be valuable for the design of parasite-specific inhibitors.