The Experts below are selected from a list of 3303 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, Antonella Pasquato, Nabil G. Seidah, Giuseppe Zanotti, Laura Cendron, 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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Arenavirus nucleoprotein targets interferon regulatory factor activating kinase ikke
Journal of Virology, 2012Co-Authors: Christelle Pythoud, Juan Carlos De La Torre, Luis Martinezsobrido, W Shanaka W I Rodrigo, Giulia Pasqual, Sylvia Rothenberger, Stefan KunzAbstract: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 IKKe but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKe interaction was highly conserved among Arenaviruses from different clades. In LCMV-infected cells, IKKe colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKe (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKe as a novel target of Arenavirus NP. Engagement of NP seems to sequester IKKe in an inactive complex. Considering the important functions of IKKe in innate antiviral immunity and other cellular processes, the NP-IKKe interaction likely plays a crucial role in Arenavirus-host interaction.
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molecular characterization of the processing of Arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme 1 site 1 protease
Journal of Virology, 2012Co-Authors: Dominique J Burri, Antonella Pasquato, Nabil G. Seidah, Giulia Pasqual, Cylia Rochat, Stefan KunzAbstract: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.
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antiviral activity of a small molecule inhibitor of Arenavirus glycoprotein processing by the cellular site 1 protease
Journal of Virology, 2011Co-Authors: Shuzo Urata, Antonella Pasquato, Stefan Kunz, Nadezhda E. Yun, Slobodan Paessler, Juan Carlos De La TorreAbstract: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.
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pathogenesis of Arenavirus hemorrhagic fevers
Expert Review of Anti-infective Therapy, 2011Co-Authors: Marielaurence Moraz, Stefan KunzAbstract:Viral hemorrhagic fevers (VHFs) caused by Arenaviruses belong to the most devastating emerging human diseases and represent serious public health problems. Arenavirus VHFs in humans are acute diseases characterized by fever and, in severe cases, different degrees of hemorrhages associated with a shock syndrome in the terminal stage. Over the past years, much has been learned about the pathogenesis of Arenaviruses at the cellular level, in particular their ability to subvert the host cell's innate antiviral defenses. Clinical studies and novel animal models have provided important new information about the interaction of hemorrhagic Arenaviruses with the host's adaptive immune system, in particular virus-induced immunosuppression, and have provided the first hints towards an understanding of the terminal hemorrhagic shock syndrome. The scope of this article is to review our current knowledge on Arenavirus VHF pathogenesis with an emphasis on recent developments.
Juan Carlos De La Torre - One of the best experts on this subject based on the ideXlab platform.
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development of live attenuated Arenavirus vaccines based on codon deoptimization of the viral glycoprotein
Virology, 2017Co-Authors: Benson Yee Hin Cheng, Juan Carlos De La Torre, Aitor Nogales, Luis MartinezsobridoAbstract:Several Arenaviruses, chiefly Lassa (LASV) in West Africa, cause hemorrhagic fever (HF) disease in humans and pose important public health problems in their endemic regions. To date, there are no FDA-approved Arenavirus vaccines and current anti-arenaviral therapy is limited to the use of ribavirin that has very limited efficacy. In this work we document that a recombinant prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) with a codon deoptimized (CD) surface glycoprotein (GP), rLCMV/CD, exhibited wild type (WT)-like growth properties in cultured cells despite barely detectable GP expression levels in rLCMV/CD-infected cells. Importantly, rLCMV/CD was highly attenuated in vivo but able to induce complete protection against a subsequent lethal challenge with rLCMV/WT. Our findings support the feasibility of implementing an Arenavirus GP CD-based approach for the development of safe and effective live-attenuated vaccines (LAVs) to combat diseases caused by human pathogenic Arenaviruses.
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identification and mechanism of action of a novel small molecule inhibitor of Arenavirus multiplication
Journal of Virology, 2015Co-Authors: Nhi Ngo, Beatrice Cubitt, Masaharu Iwasaki, Juan Carlos De La TorreAbstract:UNLABELLED Several Arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed Arenavirus vaccines and current antiArenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406. IMPORTANCE Hemorrhagic fever Arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by Arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed Arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors Arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.
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General Molecular Strategy for Development of Arenavirus Live-Attenuated Vaccines.
Journal of virology, 2015Co-Authors: Masaharu Iwasaki, Beatrice Cubitt, Nhi Ngo, John R. Teijaro, Juan Carlos De La TorreAbstract:ABSTRACT Hemorrhagic fever Arenaviruses (HFA) pose important public health problems in regions where they are endemic. Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa, causing a large number of Lassa fever cases associated with high morbidity and mortality. Concerns about human-pathogenic Arenaviruses are exacerbated because of the lack of FDA-licensed Arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. The Mopeia virus (MOPV)/LASV reassortant (ML29) is a LASV candidate live-attenuated vaccine (LAV) that has shown promising results in animal models. Nevertheless, the mechanism of ML29 attenuation remains unknown, which raises concerns about the phenotypic stability of ML29 in response to additional mutations. Development of LAVs based on well-defined molecular mechanisms of attenuation will represent a major step in combatting HFA. We used the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) to develop a general molecular strategy for Arenavirus attenuation. Our approach involved replacement of the noncoding intergenic region (IGR) of the L genome segment with the IGR of the S genome segment to generate a recombinant LCMV, rLCMV(IGR/S-S), that was highly attenuated in vivo but induced protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV(IGR/S-S) was associated with a stable reorganization of the control of viral gene expression. This strategy can facilitate the rapid development of LAVs with the antigenic composition of the parental HFA and a mechanism of attenuation that minimizes concerns about increased virulence that could be caused by genetic changes in the LAV. IMPORTANCE Hemorrhagic fever Arenaviruses (HFA) cause high morbidity and mortality, and pose important public health problems in the regions where they are endemic. Implementation of live-attenuated vaccines (LAV) will represent a major step in combatting HFA. Here we have used the prototypic Arenavirus LCMV to document a general molecular strategy for Arenavirus attenuation that can facilitate the rapid development of safe and effective, as well as stable, LAV to combat HFA.
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Arenavirus genome rearrangement for the development of live attenuated vaccines
Journal of Virology, 2015Co-Authors: Benson Yee Hin Cheng, Emilio Ortizriano, Juan Carlos De La Torre, Luis MartinezsobridoAbstract:ABSTRACT Several members of the Arenaviridae family cause hemorrhagic fever disease in humans and pose serious public health problems in their geographic regions of endemicity as well as a credible biodefense threat. To date, there have been no FDA-approved Arenavirus vaccines, and current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the nucleoprotein (NP) and glycoprotein precursor (GPC) loci within the S segment of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than from the GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant trisegmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV (rLCMV) containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuated in vivo in a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of Arenavirus live attenuated vaccines based on rearrangement of their viral genome. IMPORTANCE Several Arenaviruses cause severe hemorrhagic fever in humans and also pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat Arenavirus infections and antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped to generate rLCMV/TransS. rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuated in vivo while conferring protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide a proof of concept for the rational development of safe and protective live attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic Arenavirus infections in humans.
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development of live attenuated Arenavirus vaccines based on codon deoptimization
Journal of Virology, 2015Co-Authors: Benson Yee Hin Cheng, Emilio Ortizriano, Juan Carlos De La Torre, Aitor Nogales, Luis MartinezsobridoAbstract:ABSTRACT Arenaviruses have a significant impact on public health and pose a credible biodefense threat, but the development of safe and effective Arenavirus vaccines has remained elusive, and currently, no Food and Drug Administration (FDA)-licensed Arenavirus vaccines are available. Here, we explored the use of a codon deoptimization (CD)-based approach as a novel strategy to develop live-attenuated Arenavirus vaccines. We recoded the nucleoprotein (NP) of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) with the least frequently used codons in mammalian cells, which caused lower LCMV NP expression levels in transfected cells that correlated with decreased NP activity in cell-based functional assays. We used reverse-genetics approaches to rescue a battery of recombinant LCMVs (rLCMVs) encoding CD NPs (rLCMV/NP CD ) that showed attenuated growth kinetics in vitro . Moreover, experiments using the well-characterized mouse model of LCMV infection revealed that rLCMV/NP CD1 and rLCMV/NP CD2 were highly attenuated in vivo but, upon a single immunization, conferred complete protection against a subsequent lethal challenge with wild-type (WT) recombinant LCMV (rLCMV/WT). Both rLCMV/NP CD1 and rLCMV/NP CD2 were genetically and phenotypically stable during serial passages in FDA vaccine-approved Vero cells. These results provide proof of concept of the safety, efficacy, and stability of a CD-based approach for developing live-attenuated vaccine candidates against human-pathogenic Arenaviruses. IMPORTANCE Several Arenaviruses cause severe hemorrhagic fever in humans and pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat Arenavirus infections, while antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and is associated with side effects. Here, we describe the generation of recombinant versions of the prototypic Arenavirus LCMV encoding codon-deoptimized viral nucleoproteins (rLCMV/NP CD ). We identified rLCMV/NP CD1 and rLCMV/NP CD2 to be highly attenuated in vivo but able to confer protection against a subsequent lethal challenge with wild-type LCMV. These viruses displayed an attenuated phenotype during serial amplification passages in cultured cells. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated Arenavirus vaccines.
Antonella Pasquato - One of the best experts on this subject based on the ideXlab platform.
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a molecular sensor to characterize Arenavirus envelope glycoprotein cleavage by subtilisin kexin isozyme 1 site 1 protease
Journal of Virology, 2016Co-Authors: Joel Oppliger, Joel Ramos Da Palma, Dominique J Burri, Abdelmajid Khatib, Christina F. Spiropoulou, Eric Bergeron, Antonella PasquatoAbstract: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.
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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, Antonella Pasquato, Nabil G. Seidah, Giuseppe Zanotti, Laura Cendron, 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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molecular characterization of the processing of Arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme 1 site 1 protease
Journal of Virology, 2012Co-Authors: Dominique J Burri, Antonella Pasquato, Nabil G. Seidah, Giulia Pasqual, Cylia Rochat, Stefan KunzAbstract: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.
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antiviral activity of a small molecule inhibitor of Arenavirus glycoprotein processing by the cellular site 1 protease
Journal of Virology, 2011Co-Authors: Shuzo Urata, Antonella Pasquato, Stefan Kunz, Nadezhda E. Yun, Slobodan Paessler, Juan Carlos De La TorreAbstract: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.
Dominique J Burri - One of the best experts on this subject based on the ideXlab platform.
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a molecular sensor to characterize Arenavirus envelope glycoprotein cleavage by subtilisin kexin isozyme 1 site 1 protease
Journal of Virology, 2016Co-Authors: Joel Oppliger, Joel Ramos Da Palma, Dominique J Burri, Abdelmajid Khatib, Christina F. Spiropoulou, Eric Bergeron, Antonella PasquatoAbstract: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.
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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, Antonella Pasquato, Nabil G. Seidah, Giuseppe Zanotti, Laura Cendron, 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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molecular characterization of the processing of Arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme 1 site 1 protease
Journal of Virology, 2012Co-Authors: Dominique J Burri, Antonella Pasquato, Nabil G. Seidah, Giulia Pasqual, Cylia Rochat, Stefan KunzAbstract: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.
Nabil G. Seidah - 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, Antonella Pasquato, Nabil G. Seidah, Giuseppe Zanotti, Laura Cendron, 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).
'American Society for Microbiology', 2013Co-Authors: Dj Burri, Nabil G. Seidah, Ramos Da Palma, J, Zanotti Giuseppe, Cendron Laura, Pasquato A, Kunz S.Abstract:The Arenaviruses are an important family of emerging viruses including 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 of the catalytic triad, is implicated in the molecular recognition of the aromatic "signature residue" at P7 in the GPC of the Old World Lassa virus (LASV). Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1P is crucial for efficient processing of peptides derived from Old World and Clade C New World Arenaviruses, but not Clade A and B New World Arenavirus GPCs. The data suggest that during co-evolution 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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molecular characterization of the processing of Arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme 1 site 1 protease
Journal of Virology, 2012Co-Authors: Dominique J Burri, Antonella Pasquato, Nabil G. Seidah, Giulia Pasqual, Cylia Rochat, Stefan KunzAbstract: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.