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Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID 50 ], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nnHTN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc) 4 spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nnHTN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.

Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro , which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro , showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient. IMPORTANCE Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis Thus, local-pool signal transduction operates extremely efficiently and specifically., (Copyright © 2020 Kunz et al.)

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., (Copyright © 2019 American Society for Microbiology.)

Recognition of functional receptors by viruses is a key determinant for their host range, tissue tropism, and disease potential. The highly pathogenic Lassa virus (LASV) currently represents one of the most important emerging pathogens. The major cellular receptor for LASV in human cells is the ubiquitously expressed and evolutionary highly conserved extracellular matrix receptor dystroglycan (DG). In the host, DG interacts with many cellular proteins in a tissue-specific manner. The resulting distinct supramolecular complexes likely represent the functional units for viral entry, and preexisting protein-protein interactions may critically influence DG's function in productive viral entry. Using an unbiased shotgun proteomic approach, we define the largely unknown molecular composition of DG complexes present in highly susceptible epithelial cells that represent important targets for LASV during viral transmission. We further show that the specific composition of cellular DG complexes can affect DG's function in receptor-mediated endocytosis of the virus. Under steady-state conditions, epithelial DG complexes underwent rapid turnover via an endocytic pathway that shared some characteristics with DG-mediated LASV entry. However, compared to steady-state uptake of DG, LASV entry via DG occurred faster and critically depended on additional signaling by receptor tyrosine kinases and the downstream effector p21-activating kinase. In sum, we show that the specific molecular composition of DG complexes in susceptible cells is a determinant for productive virus entry and that the pathogen can manipulate the existing DG-linked endocytic pathway. This highlights another level of complexity of virus-receptor interaction and provides possible cellular targets for therapeutic antiviral intervention. IMPORTANCE Recognition of cellular receptors allows emerging viruses to break species barriers and is an important determinant for their disease potential. Many virus receptors have complex tissue-specific interactomes, and preexisting protein-protein interactions may influence their function. Combining shotgun proteomics with a biochemical approach, we characterize the molecular composition of the functional receptor complexes used by the highly pathogenic Lassa virus (LASV) to invade susceptible human cells. We show that the specific composition of the receptor complexes affects productive entry of the virus, providing proof-of-concept. In uninfected cells, these functional receptor complexes undergo dynamic turnover involving an endocytic pathway that shares some characteristics with viral entry. However, steady-state receptor uptake and virus endocytosis critically differ in kinetics and underlying signaling, indicating that the pathogen can manipulate the receptor complex according to its needs. Our study highlights a remarkable complexity of LASV-receptor interaction and identifies possible targets for therapeutic antiviral intervention., (Copyright © 2019 Herrador et al.)

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., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2018 American Society for Microbiology.)

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., (Copyright © 2017 American Society for Microbiology.)

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.

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.

Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728-7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cis-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.

A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P. Treatment with the S1P inhibitor dec-RRLL-CMK resulted in specific blocking of viral spread and virus production of LCMV. Combination of the protease inhibitor with ribavirin, currently used clinically for treatment of human arenavirus infections, resulted in additive drug effects. In cells deficient in S1P, the furin-dependent LCMV variant established persistent infection, whereas wild-type LCMV underwent extinction without the emergence of S1P-independent escape variants. Together, the potent antiviral activity of an inhibitor of S1P-dependent GPC cleavage, the additive antiviral effect with ribavirin, and the low probability of emergence of S1P-independent viral escape variants make S1P-mediated GPC processing by peptide-derived inhibitors a promising strategy for the development of novel antiarenaviral drugs.

After infection with RML murine scrapie agent, transgenic (tg) mice expressing prion protein (PrP) without its glycophosphatidylinositol (GPI) membrane anchor (GPI(-/-) PrP tg mice) continue to make abundant amounts of the abnormally folded disease-associated PrPres but have a normal life span. In contrast, all age-, sex-, and genetically matched mice with a GPI-anchored PrP become moribund and die due to a chronic progressive neurodegenerative disease by 160 days after RML scrapie agent infection. We report here that infected GPI(-/-) PrP tg mice, although free from progressive neurodegenerative disease of the cerebellum and extrapyramidal and pyramidal systems, nevertheless suffer defects in learning and memory, long-term potentiation, and neuronal excitability. Such dysfunction increases over time and is associated with an increase in gamma aminobutyric acid (GABA) inhibition but not loss of excitatory glutamate/N-methyl-d-aspartic acid. Enhanced deposition of abnormally folded infectious PrP (PrPsc or PrPres) in the central nervous system (CNS) localizes with GABAA receptors. This occurs with minimal evidence of CNS spongiosis or apoptosis of neurons. The use of monoclonal antibodies reveals an association of PrPres with GABAA receptors. Thus, the clinical defects of learning and memory loss in vivo in GPI(-/-) PrP tg mice infected with scrapie agent may likely involve the GABAergic pathway.

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.

The cellular proprotein convertase site 1 protease (S1P) has been implicated in the proteolytic processing of the glycoproteins (GPs) of Old World arenaviruses. Here we report that S1P is also involved in the processing of the GPs of the genetically more-distant South American hemorrhagic fever viruses Guanarito, Machupo, and Junin. Efficient cleavage of Guanarito virus GP, whose protease recognition sites deviate from the reported S1P consensus sequence, indicates a broader specificity of S1P than anticipated. Lack of GP processing of Junin virus dramatically reduced production of infectious virus and prevented cell-to-cell propagation. Infection of S1P-deficient cells resulted in viral persistence over several weeks without the emergence of escape variants able to use other cellular proteases for GP processing.

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor alpha-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.

Generation of infectious arenavirus-like particles requires the virus RING finger Z protein and surface glycoprotein precursor (GPC) and the correct processing of GPC into GP1, GP2, and a stable signal peptide (SSP). Z is the driving force of arenavirus budding, whereas the GP complex (GPc), consisting of hetero-oligomers of SSP, GP1, and GP2, forms the viral envelope spikes that mediate receptor recognition and cell entry. Based on the roles played by Z and GP in the arenavirus life cycle, we hypothesized that Z and the GPc should interact in a manner required for virion formation. Here, using confocal microscopy and coimmunoprecipitation assays, we provide evidence for subcellular colocalization and biochemical interaction, respectively, of Z and the GPc. Our results from mutation-function analysis reveal that Z myristoylation, but not the Z late (L) or RING domain, is required for Z-GPc interaction. Moreover, Z interacted directly with SSP in the absence of other components of the GPc. We obtained similar results with Z and GPC from the prototypical arenavirus lymphocytic choriomeningitis virus and the hemorrhagic fever arenavirus Lassa fever virus.

alpha-Dystroglycan (DG) is an important cellular receptor for extracellular matrix (ECM) proteins and also serves as the receptor for Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) and clade C New World arenaviruses. In the host cell, alpha-DG is subject to a remarkably complex pattern of O glycosylation that is crucial for its interactions with ECM proteins. Two of these unusual sugar modifications, protein O mannosylation and glycan modifications involving the putative glycosyltransferase LARGE, have recently been implicated in arenavirus binding. Considering the complexity of alpha-DG O glycosylation, our present study was aimed at the identification of the specific O-linked glycans on alpha-DG that are recognized by arenaviruses. As previously shown for LCMV, we found that protein O mannosylation of alpha-DG is crucial for the binding of arenaviruses of distinct phylogenetic origins, including LFV, Mobala virus, and clade C New World arenaviruses. In contrast to the highly conserved requirement for O mannosylation, more generic O glycans present on alpha-DG are dispensable for arenavirus binding. Despite the critical role of O-mannosyl glycans for arenavirus binding under normal conditions, the overexpression of LARGE in cells deficient in O mannosylation resulted in highly glycosylated alpha-DG that was functional as a receptor for arenaviruses. Thus, modifications by LARGE but not O-mannosyl glycans themselves are most likely the crucial structures recognized by arenaviruses. Together, the data demonstrate that arenaviruses recognize the same highly conserved O-glycan structures on alpha-DG involved in ECM protein binding, indicating a strikingly similar mechanism of receptor recognition by pathogen- and host-derived ligands.

Neonatal infection of most mouse strains with lymphocytic choriomeningitis virus (LCMV) leads to a life-long persistent infection characterized by high virus loads in the central nervous system (CNS) in the absence of inflammation and tissue destruction. These mice, however, exhibit impaired learning and memory. The occurrence of cognitive defects in the absence of overt CNS pathology led us to the hypothesis that chronic virus infection may contribute to neuronal dysfunction by altering the host's gene expression profile. To test this hypothesis, we examined the impact of LCMV persistence on host gene expression in the CNS. To model the natural route of human congenital CNS infection observed with a variety of viruses, we established a persistently infected mouse colony where the virus was maintained via vertical transmission from infected mothers to offspring (LCMV-cgPi). LCMV-cgPi mice exhibited a lifelong persistent infection involving the CNS; the infection was associated with impaired spatial-temporal learning. Despite high viral loads in neurons of the brains of adult LCMV-cgPi mice, we detected changes in the host's CNS gene expression for only 75 genes, 56 and 19 being significantly induced and reduced, respectively. The majority of the genes induced in the brain of LCMV-cgPi mice were interferon (IFN)-stimulated genes (ISGs) and included the transcription factors STAT1 and IRF9, the ISG15 protease UBP43, and the glucocorticoid attenuated-response genes GARG16 and GARG49. Based on their crucial role in antiviral defense, these ISGs may play an important role in limiting viral spread and replication. However, since IFNs have also been implicated in adverse effects on neuronal function, the chronic induction of some ISGs may also contribute to the observed cognitive impairment.

The receptor for lymphocytic choriomeningitis virus (LCMV), the human pathogenic Lassa fever virus (LFV), and clade C New World arenaviruses is alpha-dystroglycan (alpha-DG), a cell surface receptor for proteins of the extracellular matrix (ECM). Specific posttranslational modification of alpha-DG by the glycosyltransferase LARGE is critical for its function as an ECM receptor. In the present study, we show that LARGE-dependent modification is also crucial for alpha-DG's function as a cellular receptor for arenaviruses. Virus binding involves the mucin-type domain of alpha-DG and depends on modification by LARGE. A crucial role of the LARGE-dependent glycosylation of alpha-DG for virus binding is found for several isolates of LCMV, LFV, and the arenaviruses Mobala and Oliveros. Since the posttranslational modification by LARGE is crucial for alpha-DG recognition by both arenaviruses and the host-derived ligand laminin, it also influences competition between virus and laminin for alpha-DG. Hence, LARGE-dependent glycosylation of alpha-DG has important implications for the virus-host cell interaction and the pathogenesis of LFV in humans.

The cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as alpha-dystroglycan (alpha-DG), a cell surface receptor that provides a molecular link between the extracellular matrix and the actin-based cytoskeleton. In the present study, we show that LFV binds to alpha-DG with high affinity in the low-nanomolar range. Recombinant vesicular stomatitis virus pseudotyped with LFV glycoprotein (GP) adopted the receptor binding characteristics of LFV and depended on alpha-DG for infection of cells. Mapping of the binding site of LFV on alpha-DG revealed that LFV binding required the same domains of alpha-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin alpha1 and alpha2 chains for alpha-DG binding. Together with our previous studies on receptor binding of the prototypic immunosuppressive LCMV isolate LCMV clone 13, these findings indicate a high degree of conservation in the receptor binding characteristics between the highly human-pathogenic LFV and murine-immunosuppressive LCMV isolates.

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.

alpha-Dystroglycan (alpha-DG) was recently identified as a receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses, including Lassa fever virus (W. Cao, M. D. Henry, P. Borrow, H. Yamada, J. H. Elder, E. V. Ravkov, S. T. Nichol, R. W. Compans, K. P. Campbell, and M. B. A. Oldstone, Science 282:2079-2081, 1998). Data presented in this paper indicate that the affinity of binding of LCMV to alpha-DG determines viral tropism and the outcome of infection in mice. To characterize this relationship, we evaluated the interaction between alpha-DG and several LCMV strains, variants, and reassortants. These viruses could be divided into two groups with respect to affinity of binding to alpha-DG, dependence on this protein for cell entry, viral tropism, and disease course. Viruses that exhibited high-affinity binding to alpha-DG displayed a marked dependence on alpha-DG for cell entry and were blocked from infecting mouse 3T6 fibroblasts by 1 to 4 nM soluble alpha-DG. In addition, high-affinity binding to alpha-DG correlated with an ability to infiltrate the white pulp (T-dependent) area of the spleen, cause ablation of the cytotoxic T-lymphocyte (CTL) response by day 7 postinfection, and establish a persistent infection. In contrast, viruses with a lower affinity of binding to alpha-DG were only partially inhibited from infecting alpha-DG(-/-) embryonic stem cells and required a concentration of soluble alpha-DG higher than 100 nM to prevent infection of mouse 3T6 fibroblasts. These viruses that bound at low affinity were mainly restricted to the splenic red pulp, and the host generated an effective CTL response that rapidly cleared the infection. Reassortants of viruses that bound to alpha-DG at high and low affinities were used to map genes responsible for the differences described to the S RNA, containing the virus attachment protein glycoprotein 1.