Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease

Palma, Joel Ramos da; Burri, Dominique J.; Oppliger, Joël; Salamina, Marco; Cendron, Laura; Laureto, Patrizia Polverino de; Seidah, Nabil G.; Kunz, Stefan; Pasquato, Antonella

doi:10.1074/jbc.m114.588525

Cited by 23 publications

(66 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…An expression plasmid containing the full-length cDNA of LUJV was provided by the Viral Special Pathogens Branch of the U.S. Centers for Disease Control and Prevention, Atlanta, GA. The SKI-1/S1P sensors containing the 9-mer recognition sequence of LASV GPC, IYISRRLL2G, and the uncleavable sequence (U-LASV) IYISEELL2G have been described in a previous report (17). Sensors containing 9-mer peptides derived from arenavirus GPCs were generated by the insertion of specific oligonucleotides coding for the designed sequence flanked by unique restriction sites at the 3= and 5= ends, as reported previously (17).…”

Section: Methodsmentioning

confidence: 99%

“…The SKI-1/S1P sensors containing the 9-mer recognition sequence of LASV GPC, IYISRRLL2G, and the uncleavable sequence (U-LASV) IYISEELL2G have been described in a previous report (17). Sensors containing 9-mer peptides derived from arenavirus GPCs were generated by the insertion of specific oligonucleotides coding for the designed sequence flanked by unique restriction sites at the 3= and 5= ends, as reported previously (17). This cassette insertion method allows for the rapid generation of constructs containing different cleavable motifs.…”

Section: Methodsmentioning

confidence: 99%

“…SRD12B and CHO-K1 cells were transfected with Lipofectamine as reported elsewhere (27). Sensor transfections were performed using polyethylenimine (PEI) obtained from Polysciences as described previously (17). At 4 h posttransfection, solutions were replaced by fresh medium.…”

Section: Methodsmentioning

confidence: 99%

“…Upon translocation into the endoplasmic reticulum (ER), SKI-1/S1P zymogen activation involves autocatalytic maturation by sequential cleavages of the N-terminal prodomain, first at sites B= and B (RKVF2RSLK 137 2) and then at site C (RRLL 186 2) and the newly described site C= (RRAS 166 2) (boldface and underlining highlight conserved residues) (17). The end product, SKI-1/S1P C, represents the fully mature enzyme (18,19).…”

mentioning

confidence: 99%

“…Since full-length GPCs of both LCMV and GTOV are readily cleaved in a range of mammalian cells (9,11,12), the lack of processing suggests that these otherwise powerful in vitro assays do not accurately recapitulate the authentic cellular environment. Here we employ a recently developed robust and reliable cellbased assay for the detection of endogenous SKI-1/S1P activity (17) to characterize arenavirus GPC processing in a quantitative manner. First, we show that our sensor accurately reproduces key features of SKI-1/S1P-mediated processing of authentic arenavirus GPCs, including LCMV GPC, and we define the minimal structures necessary and sufficient to confer specificity and a subcellular location of cleavage.…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Oppliger

Palma

Burri

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

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. IMPORTANCEArenaviruses 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.A renaviruses are a large and diverse family of emerging viruses that includes several causative agents of severe hemorrhagic fever in humans. The family Arenaviridae is currently classified int...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Oppliger

Palma

Burri

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

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

Löw,

Möller,

Stegmann

et al. 2023

Journal of Medical Virology

Self Cite

View full text Add to dashboard Cite

The emerging viruses SARS‐CoV‐2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS‐CoV‐2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme‐1/site‐1 protease (SKI‐1/S1P), respectively. We report improved luciferase‐based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high‐throughput manner. We identified 23 FDA‐approved small molecules, among them monensin which displayed broad activity against furin and SKI‐1/S1P. Monensin inhibited arenaviruses and SARS‐CoV‐2 in a dose‐dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS‐CoV‐2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS‐CoV‐2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad‐spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

show abstract

Cleavage of the Glycoprotein of Arenaviruses

Pasquato

Cendron

Kunz

2018

Activation of Viruses by Host Proteases

View full text Add to dashboard Cite

the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

show abstract

Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease

Cited by 23 publications

References 38 publications

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

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

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

Cleavage of the Glycoprotein of Arenaviruses

Contact Info

Product

Resources

About