The Experts below are selected from a list of 267 Experts worldwide ranked by ideXlab platform
Stefan Kunz - One of the best experts on this subject based on the ideXlab platform.
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differential recognition of old World and New World Arenavirus envelope glycoproteins by subtilisin kexin isozyme 1 ski 1 site 1 protease s1p
Journal of Virology, 2013Co-Authors: Dominique J. Burri, Joel Ramos Da Palma, Nabil G. Seidah, Giuseppe Zanotti, Laura Cendron, Antonella Pasquato, Stefan KunzAbstract:ABSTRACT 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 residueY285 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.
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Differential Recognition of Old World and New World Arenavirus Envelope Glycoproteins by Subtilisin Kexin Isozyme 1 (SKI-1)/Site 1 Protease (S1P)
Journal of virology, 2013Co-Authors: Dominique J. Burri, Joel Ramos Da Palma, Nabil G. Seidah, Giuseppe Zanotti, Laura Cendron, Antonella Pasquato, Stefan KunzAbstract:ABSTRACT 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 residueY285 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.
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The role of the vascular endothelium in Arenavirus haemorrhagic fevers
Thrombosis and haemostasis, 2009Co-Authors: Stefan KunzAbstract:Viral haemorrhagic fevers (VHF) caused by Arenaviruses are among the most devastating emerging human diseases. The most important pathogen among the Arenaviruses is Lassa virus (LASV), the causative agent of Lassa fever that is endemic to West Africa. On the South American continent, the New World Arenavirus Junin virus (JUNV), Machupo (MACV), Guanarito (GTOV), and Sabia virus (SABV) have emerged as causative agents of severe VHFs. Clinical and experimental studies on Arenavirus VHF have revealed a crucial role of the endothelium in their pathogenesis. However, in contrast to other VHFs, haemorrhages are not a salient feature of Lassa fever and fatal cases do not show overt destruction of vascular tissue. The functional alteration of the vascular endothelium that precede shock and death in fatal Lassa fever may be due to more subtle direct or indirect effects of the virus on endothelial cells. Haemorrhagic disease manifestations and vascular involvement are more pronounced in the VHF caused by the South American haemorrhagic fever viruses. Recent studies on JUNV revealed perturbation of specific endothelial cell function, including expression of cell adhesion molecules, coagulation factors, and vasoactive mediators as a consequence of productive viral infection. These studies provided first possible links to some of the vascular abnormalities observed in patients, however, their relevance in vivo remains to be investigated.
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Different Mechanisms of Cell Entry by Human-Pathogenic Old World and New World Arenaviruses
Journal of virology, 2008Co-Authors: Jillian M. Rojek, Juan Carlos De La Torre, Ana B. Sanchez, Ngoc Thao Nguyen, Stefan KunzAbstract: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.
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Cell entry by human pathogenic Arenaviruses.
Cellular microbiology, 2007Co-Authors: Stefan KunzAbstract:The Arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15-35%. The present review focuses on the first steps of infection with human pathogenic Arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World Arenaviruses these pathogens use different primary receptors. The Old World Arenaviruses employ alpha-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World Arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World Arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World Arenavirus lymphocytic choriomeningitis virus. Upon internalization, Arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While Arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for Arenaviruses with important implications for viral entry.
Slobodan Paessler - One of the best experts on this subject based on the ideXlab platform.
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A single mutation (V64G) within the RING Domain of Z attenuates Junin virus.
PLOS Neglected Tropical Diseases, 2020Co-Authors: Steven Hallam, John T. Manning, Junki Maruyama, Alexey Seregin, Cheng Huang, David H. Walker, Juan Carlos De La Torre, Slobodan PaesslerAbstract:Junin virus (JUNV) is a New World Arenavirus that is the causative agent of Argentine hemorrhagic fever (AHF). Candid#1 (Can) is a live-attenuated vaccine strain of JUNV that since its introduction has resulted in a marked decrease in AHF incidence within the endemic regions of the Pampas in Argentina. Originally, the viral determinants and mechanisms of Can attenuation were not well understood. Recent work has identified the glycoprotein as the major attenuating factor for Can. The establishment of attenuating strategies based on any of the other viral proteins, however, has not been pursued. Here, we document the role of Can Z resulting in incompatibilities with wild type JUNV that results in decreased growth in vitro. In addition, this incompatibility results in attenuation of the virus in the guinea pig model. Further, we identify a single mutation (V64G) in the Z protein that is able to confer this demonstrated attenuation. By establishing and characterizing a novel attenuation strategy for New World mammArenaviruses, we hope to aid future vaccine development for related emerging pathogens including Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV).
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Monoclonal Antibodies with Neutralizing Activity and Fc-Effector Functions against the Machupo Virus Glycoprotein.
Journal of virology, 2020Co-Authors: Fatima Amanat, Cheng Huang, Slobodan Paessler, James Duehr, Gene S. Tan, Florian KrammerAbstract:Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever (BHF), is a New World Arenavirus that was first isolated in Bolivia from a human spleen in 1963. Due to the lack of a specific vaccine or therapy, this virus is considered a major risk to public health and is classified as a category A priority pathogen by the U.S. National Institutes of Health. In this study, we used DNA vaccination against the MACV glycoprotein precursor complex (GPC) and murine hybridoma technology to generate 25 mouse monoclonal antibodies (MAbs) against the GPC of MACV. Out of 25 MAbs, five were found to have potent neutralization activity in vitro against a recombinant vesicular stomatitis virus expressing MACV GPC (VSV-MACV) as well as against authentic MACV. Furthermore, the five neutralizing MAbs exhibited strong antibody-dependent cellular cytotoxicity (ADCC) activity in a reporter assay. When tested in vivo using VSV-MACV in a Stat2-/- mouse model, three MAbs significantly lowered viral loads in the spleen. Our work provides valuable insights into epitopes targeted by neutralizing antibodies that could be potent targets for vaccines and therapeutics and shed light on the importance of effector functions in immunity against MACV.IMPORTANCE MACV infections are a significant public health concern and lead to high case fatality rates. No specific treatment or vaccine for MACV infections exist. However, cases of Junin virus infection, a related virus, can be treated with convalescent-phase serum. This indicates that a MAb-based therapy for MACV could be effective. Here, we describe several MAbs that neutralize MACV and could be used for this purpose.
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highly pathogenic New World Arenavirus infection activates the pattern recognition receptor protein kinase r without attenuating virus replication in human cells
Journal of Virology, 2017Co-Authors: Cheng Huang, Takaaki Koma, Olga A. Kolokoltsova, Elizabeth J Mateer, Slobodan PaesslerAbstract:The Arenavirus family consists of several highly pathogenic viruses, including the Old World (OW) Arenavirus Lassa fever virus (LASV) and the New World (NW) Junin virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic Arenaviruses is distinct in many aspects. JUNV and MACV infections readily induce an interferon (IFN) response in human cells, while LASV infection usually triggers an undetectable or weak IFN response. JUNV induces an IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs (vRNAs) during infection and activates IFN response. Double-stranded-RNA (dsRNA)-activated protein kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Here we report that infection with NW Arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor α subunit of eukaryotic initiation factor 2 (eIF2α). Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired but was slightly augmented in wild-type (wt) cells compared to that in PKR-deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV- or MACV-infected PKR-deficient cells, which was inversely correlated with virus replication.IMPORTANCE The detection of viral RNA by host non-self RNA sensors, including RIG-I and MDA5, is critical to the initiation of the innate immune response to RNA virus infection. Among pathogenic Arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW Arenaviruses JUNV and MACV readily trigger an IFN response in a RIG-I-dependent manner. Here, we demonstrate for the first time that pathogenic NW Arenaviruses JUNV and MACV, but not the OW Arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor, during infection. Interestingly, the replication of JUNV and MACV was not restricted but was rather slightly augmented in the presence of PKR. Our data provide New evidence for a distinct interplay between host non-self RNA sensors and pathogenic Arenaviruses, which also provides insights into the pathogenesis of Arenaviruses and may facilitate the design of vaccines and treatments against Arenavirus-caused diseases.
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Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression.
Frontiers in cellular and infection microbiology, 2017Co-Authors: John T. Manning, Alexey Seregin, Cheng Huang, Juan Carlos De La Torre, Takaaki Koma, Nadezhda E Yun, José M. Barral, Slobodan PaesslerAbstract:Junin virus (JUNV), a highly pathogenic New World Arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166 to 168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.
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the ectodomain of glycoprotein from the candid 1 vaccine strain of junin virus rendered machupo virus partially attenuated in mice lacking ifn αβ γ receptor
PLOS Neglected Tropical Diseases, 2016Co-Authors: Takaaki Koma, Cheng Huang, Judith F. Aronson, Aida G Walker, Milagros Miller, Jeanon N Smith, Michael Patterson, Slobodan PaesslerAbstract:Machupo virus (MACV), a New World Arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC.
Juan Carlos De La Torre - One of the best experts on this subject based on the ideXlab platform.
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A single mutation (V64G) within the RING Domain of Z attenuates Junin virus.
PLOS Neglected Tropical Diseases, 2020Co-Authors: Steven Hallam, John T. Manning, Junki Maruyama, Alexey Seregin, Cheng Huang, David H. Walker, Juan Carlos De La Torre, Slobodan PaesslerAbstract:Junin virus (JUNV) is a New World Arenavirus that is the causative agent of Argentine hemorrhagic fever (AHF). Candid#1 (Can) is a live-attenuated vaccine strain of JUNV that since its introduction has resulted in a marked decrease in AHF incidence within the endemic regions of the Pampas in Argentina. Originally, the viral determinants and mechanisms of Can attenuation were not well understood. Recent work has identified the glycoprotein as the major attenuating factor for Can. The establishment of attenuating strategies based on any of the other viral proteins, however, has not been pursued. Here, we document the role of Can Z resulting in incompatibilities with wild type JUNV that results in decreased growth in vitro. In addition, this incompatibility results in attenuation of the virus in the guinea pig model. Further, we identify a single mutation (V64G) in the Z protein that is able to confer this demonstrated attenuation. By establishing and characterizing a novel attenuation strategy for New World mammArenaviruses, we hope to aid future vaccine development for related emerging pathogens including Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV).
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Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression.
Frontiers in cellular and infection microbiology, 2017Co-Authors: John T. Manning, Alexey Seregin, Cheng Huang, Juan Carlos De La Torre, Takaaki Koma, Nadezhda E Yun, José M. Barral, Slobodan PaesslerAbstract:Junin virus (JUNV), a highly pathogenic New World Arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166 to 168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.
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the glycoprotein precursor gene of junin virus determines the virulence of the romero strain and the attenuation of the candid 1 strain in a representative animal model of argentine hemorrhagic fever
Journal of Virology, 2015Co-Authors: Alexey Seregin, John T. Manning, Cheng Huang, Judith F. Aronson, Aida G Walker, Nadezhda E Yun, Milagros Miller, Jeanon N Smith, Jennifer K Smith, Juan Carlos De La TorreAbstract:ABSTRACT The New World Arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 (Can) strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective, and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV (rJUNV) strains encoding different gene combinations of the pathogenic Romero (Rom) and attenuated Can strains of JUNV. All strains of rJUNV exhibited in vitro growth kinetics similar to those of their parental counterparts. Analysis of virulence of the rJUNV in a guinea pig model of lethal infection that closely reproduces the features of AHF identified the envelope glycoproteins (GPs) as the major determinants of pathogenesis and attenuation of JUNV. Accordingly, rJUNV strains expressing the full-length GPs of Rom and Can exhibited virulent and attenuated phenotypes, respectively, in guinea pigs. Mutation F427I in the transmembrane region of JUNV envelope glycoprotein GP2 has been shown to attenuate the neurovirulence of JUNV in suckling mice. We document that in the guinea pig model of AHF, mutation F427I in GP2 is also highly attenuating but insufficient to prevent virus dissemination and development of mild clinical and pathological symptoms, indicating that complete attenuation of JUNV requires additional mutations present in Can glycoprotein precursor (GPC). IMPORTANCE Development of antiviral strategies against viral hemorrhagic fevers, including AHF, is one of the top priorities within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Live-attenuated Candid #1 strain, derived from the 44th mouse brain passage of the prototype XJ strain of JUNV, has been demonstrated to be safe, immunogenic, and highly protective and is currently licensed for human use in Argentina. However, the bases for the attenuated phenotype of Candid #1 have not been established. Therefore, the identification and functional characterization of viral genetic factors implicated in JUNV pathogenesis and attenuation would significantly improve the understanding of the molecular mechanisms underlying AHF and facilitate the development of novel antiviral strategies.
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Arenavirus reverse genetics for vaccine development.
Journal of General Virology, 2013Co-Authors: Emilio Ortiz-riaño, Juan Carlos De La Torre, Benson Yee Hin Cheng, Luis Martínez-sobridoAbstract:Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in Arenavirus research. However, rescue of recombinant Arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant Arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for Arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic Arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World Arenavirus Junin. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1.
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rescue from cloned cdnas and in vivo characterization of recombinant pathogenic romero and live attenuated candid 1 strains of junin virus the causative agent of argentine hemorrhagic fever disease
Journal of Virology, 2011Co-Authors: Sébastien Emonet, Alexey Seregin, Juan Carlos De La Torre, Aida G Walker, Nadezhda E Yun, Allison Poussard, Slobodan PaesslerAbstract:The New World Arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), which is associated with high morbidity and significant mortality. Several pathogenic strains of JUNV have been documented, and a highly attenuated vaccine strain (Candid #1) was generated and used to vaccinate the human population at risk. The identification and functional characterization of viral genetic determinants associated with AHF and Candid #1 attenuation would contribute to the elucidation of the mechanisms contributing to AHF and the development of better vaccines and therapeutics. To this end, we used reverse genetics to rescue the pathogenic Romero and the attenuated Candid #1 strains of JUNV from cloned cDNAs. Both recombinant Candid #1 (rCandid #1) and Romero (rRomero) had the same growth properties and phenotypic features in cultured cells and in vivo as their corresponding parental viruses. Infection with rRomero caused 100% lethality in guinea pigs, whereas rCandid #1 infection was asymptomatic and provided protection against a lethal challenge with Romero. Notably, Romero and Candid #1 trans-acting proteins, L and NP, required for virus RNA replication and gene expression were exchangeable in a minigenome rescue assay. These findings support the feasibility of studies aimed at determining the contribution of each viral gene to JUNV pathogenesis and attenuation. In addition, we rescued Candid #1 viruses with three segments that efficiently expressed foreign genes introduced into their genomes. This finding opens the way for the development of a safe multivalent Arenavirus vaccine.
Susan R. Ross - One of the best experts on this subject based on the ideXlab platform.
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CACNA1S haploinsufficiency confers resistance to New World Arenavirus infection.
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Nicolás Sarute, Susan R. RossAbstract:Understanding the genetics of susceptibility to infectious agents is of great importance to our ability to combat disease. Here, we show that voltage-gated calcium channels (VGCCs) are critical for cellular binding and entry of the New World Arenaviruses Junin and Tacaribe virus, suggesting that zoonosis via these receptors could occur. Moreover, we demonstrate that α1s haploinsufficiency renders cells and mice more resistant to infection by these viruses. In addition to being more resistant to infection, haploinsufficient cells and mice required a lower dosage of VGCC antagonists to block infection. These studies underscore the importance of genetic variation in susceptibility to both viruses and pharmaceutics.
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TRIM2, a novel member of the antiviral family, limits New World Arenavirus entry
2019Co-Authors: Nicolás Sarute, Madakasira Lavanya, Nouhou Ibrahim, Bani Medegan Fagla, Christian Cuevas, Spyridon Stavrou, Guliz Otkiran-clare, Henna Tyynismaa, Jorge Henao-mejia, Susan R. RossAbstract:Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot–Marie–Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World Arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap.
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New World Arenavirus Biology.
Annual review of virology, 2017Co-Authors: Nicolás Sarute, Susan R. RossAbstract:Hemorrhagic fevers caused by viruses were identified in the late 1950s in South America. These viruses have existed in their hosts, the New World rodents, for millions of years. Their emergence as infectious agents in humans coincided with changes in the environment and farming practices that caused explosions in their host rodent populations. Zoonosis into humans likely occurs because the pathogenic New World Arenaviruses use human transferrin receptor 1 to enter cells. The mortality rate after infection with these viruses is high, but the mechanism by which disease is induced is still not clear. Possibilities include direct effects of cellular infection or the induction of high levels of cytokines by infected sentinel cells of the immune system, leading to endothelia and thrombocyte dysfunction and neurological disease. Here we provide a review of the ecology and molecular and cellular biology of New World Arenaviruses, as well as a discussion of the current animal models of infection. The development of animal models, coupled with an improved understanding of the infection pathway and host response, should lead to the discovery of New drugs for treating infections.
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siRNA Screen for Genes That Affect Junín Virus Entry Uncovers Voltage-Gated Calcium Channels as a Therapeutic Target
Science translational medicine, 2013Co-Authors: Madakasira Lavanya, Christian D. Cuevas, Monica Thomas, Sara Cherry, Susan R. RossAbstract:New World hemorrhagic fever Arenavirus infection results in 15 to 30% mortality in humans. We performed a high-throughput small interfering RNA screen with Junin virus glycoprotein-pseudotyped viruses to find potential host therapeutic targets. Voltage-gated calcium channel (VGCC) subunits, for which there are Food and Drug Administration (FDA)–approved drugs, were identified in the screen. Knockdown of VGCC subunits or treatment with channel blockers diminished Junin virus–cell fusion and entry into cells and thereby decreased infection. Gabapentin, an FDA-approved drug used to treat neuropathic pain that targets the α 2 δ 2 subunit, inhibited infection of mice by the Candid 1 vaccine strain of the virus. These findings demonstrate that VGCCs play a role in virus infection and have the potential to lead to therapeutic intervention of New World Arenavirus infection.
Michael Farzan - One of the best experts on this subject based on the ideXlab platform.
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TIM-family Proteins Promote Infection of Multiple Enveloped Viruses through Virion-associated Phosphatidylserine
PLoS pathogens, 2013Co-Authors: Stephanie Jemielity, Michael Farzan, Jinyize J. Wang, Ying Kai Chan, Asim A. Ahmed, Sheena Monahan, Gordon J. Freeman, Dale T. UmetsuAbstract:Human T-cell Immunoglobulin and Mucin-domain containing proteins (TIM1, 3, and 4) specifically bind phosphatidylserine (PS). TIM1 has been proposed to serve as a cellular receptor for hepatitis A virus and Ebola virus and as an entry factor for dengue virus. Here we show that TIM1 promotes infection of retroviruses and virus-like particles (VLPs) pseudotyped with a range of viral entry proteins, in particular those from the filovirus, flavivirus, New World Arenavirus and alphavirus families. TIM1 also robustly enhanced the infection of replication-competent viruses from the same families, including dengue, Tacaribe, Sindbis and Ross River viruses. All interactions between TIM1 and pseudoviruses or VLPs were PS-mediated, as demonstrated with liposome blocking and TIM1 mutagenesis experiments. In addition, other PS-binding proteins, such as Axl and TIM4, promoted infection similarly to TIM1. Finally, the blocking of PS receptors on macrophages inhibited the entry of Ebola VLPs, suggesting that PS receptors can contribute to infection in physiologically relevant cells. Notably, infection mediated by the entry proteins of Lassa fever virus, influenza A virus and SARS coronavirus was largely unaffected by TIM1 expression. Taken together our data show that TIM1 and related PS-binding proteins promote infection of diverse families of enveloped viruses, and may therefore be useful targets for broad-spectrum antiviral therapies.
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TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine. PLoS Pathog
2013Co-Authors: Stephanie Jemielity, Michael Farzan, Jinyize J. Wang, Ying Kai Chan, Asim A. Ahmed, Sheena Monahan, Gordon J. Freeman, Dale T. UmetsuAbstract:Human T-cell Immunoglobulin and Mucin-domain containing proteins (TIM1, 3, and 4) specifically bind phosphatidylserine (PS). TIM1 has been proposed to serve as a cellular receptor for hepatitis A virus and Ebola virus and as an entry factor for dengue virus. Here we show that TIM1 promotes infection of retroviruses and virus-like particles (VLPs) pseudotyped with a range of viral entry proteins, in particular those from the filovirus, flavivirus, New World Arenavirus and alphavirus families. TIM1 also robustly enhanced the infection of replication-competent viruses from the same families, including dengue, Tacaribe, Sindbis and Ross River viruses. All interactions between TIM1 and pseudoviruses or VLPs were PS-mediated, as demonstrated with liposome blocking and TIM1 mutagenesis experiments. In addition, other PS-binding proteins, such as Axl and TIM4, promoted infection similarly to TIM1. Finally, the blocking of PS receptors on macrophages inhibited the entry of Ebola VLPs, suggesting that PS receptors can contribute to infection in physiologically relevant cells. Notably, infection mediated by the entry proteins of Lassa fever virus, influenza A virus and SARS coronavirus was largely unaffected by TIM1 expression. Taken together our data show that TIM1 and related PS-binding proteins promote infection of diverse familie
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Transferrin receptor 1 in the zoonosis and pathogenesis of New World hemorrhagic fever Arenaviruses.
Current opinion in microbiology, 2011Co-Authors: Hyeryun Choe, Jonathan Abraham, Sheli R. Radoshitzky, Stephanie Jemielity, Michael FarzanAbstract:At least five New World Arenaviruses cause severe human hemorrhagic fevers. These viruses are transmitted to humans through contact with their respective South American rodent hosts. Each uses human transferrin receptor 1 (TfR1) as its obligate receptor. Accidental similarities between human TfR1 and TfR1 orthologs of arenaviral host species enable zoonoses, whereas mice and rats are not infectable because they lack these TfR1 determinants of infection. All pathogenic New World Arenaviruses bind to a common region of the apical domain of TfR1. The ability of a New World Arenavirus to use human TfR1 is absolutely predictive of its ability to cause hemorrhagic fevers in humans. Nonpathogenic Arenaviruses, closely related to hemorrhagic fever Arenaviruses, cannot utilize human TfR1 but efficiently enter cells through TfR1 orthologs of their native rodent hosts. Mutagenesis studies suggest that minor changes in the entry glycoproteins of these nonpathogenic viruses may allow human transmission. TfR1 is upregulated as a result of iron sequestration during the acute-phase response to infection, and the severity of disease may result from amplification of viral replication during this response.
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Structural basis for receptor recognition by New World hemorrhagic fever Arenaviruses
Nature Structural & Molecular Biology, 2010Co-Authors: Jonathan Abraham, Kevin D. Corbett, Michael Farzan, Hyeryun Choe, Stephen C. HarrisonAbstract:New World hemorrhagic fever Arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World Arenavirus host specificity. Analysis of sequence variation among New World Arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World Arenaviruses. We propose that New World Arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts. New World hemorrhagic fever Arenaviruses are rodent transmitted agents that cause severe, often fatal human disease. The structure of the Machupo virus glycoprotein 1 subunit in complex with its human cellular receptor, transferrin receptor 1, was solved. The interaction interfaces between the two proteins and sequence alignments suggest that these viruses fortuitously acquired the ability to bind human transferrin receptor 1 while adapting to their natural hosts.
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Structural basis for receptor recognition by New World hemorrhagic fever Arenaviruses
Nature structural & molecular biology, 2010Co-Authors: Jonathan Abraham, Kevin D. Corbett, Michael Farzan, Hyeryun Choe, Stephen C. HarrisonAbstract:New World hemorrhagic fever Arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World Arenavirus host specificity. Analysis of sequence variation among New World Arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World Arenaviruses. We propose that New World Arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.