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

Oppliger, Joël; Palma, Joel Ramos da; Burri, Dominique J.; Bergeron, Éric; Khatib, Abdel‐Majid; Spiropoulou, Christina F.; Pasquato, Antonella; Kunz, Stefan

doi:10.1128/jvi.01751-15

Cited by 12 publications

(26 citation statements)

References 42 publications

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“…The S segment encodes the viral glycoprotein precursor (GPC) and the nucleoprotein (NP). Cellular signal peptidase cleaves GPC into a stable signal peptide (SSP) and GP1/GP2, and cellular subtilase SKI-1/S1P further cleaves GP1/GP2 into the GP1 and GP2 subunits [11][12][13][14][15]. GP1, the N-terminal subunit, mediates cellular receptor binding and is also the target of host neutralizing antibodies [16].…”

Section: Introductionmentioning

confidence: 99%

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

et al. 2021

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Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

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Section: Introductionmentioning

confidence: 99%

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

et al. 2021

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“…Previously, we developed a cell‐based biosensor of the nonbasic PC SKI‐1/S1P that included an N‐terminal Gaussia luciferase (GLuc) reporter fused to a SKI‐1/S1P‐derived membrane anchor via a cleavable peptide sequence mimicking a physiological SKI‐1/S1P recognition site . Functional tests revealed that the sensor recapitulates key features of authentic substrates, including strict SKI‐1/S1P specificity and correct subcellular location of processing . Examination of a panel of SKI‐1/S1P sensors with different SKI‐1/S1P substrate‐derived sequences revealed that the cell‐based sensors accurately predicted SKI‐1/S1P cleavage efficiency of the authentic substrate .…”

Section: Resultsmentioning

confidence: 99%

“…Due to the noncanonical nature of the basic PC recognition site in BPCS , specificity of furin cleavage was a major concern. In our previous studies with the analogous cell‐based SKI‐1/S1P sensor, we found that in vitro processing of peptides derived from recognition sequences of known SKI‐1/S1P substrates sometimes failed, whereas the exact same sequence underwent efficient cleavage in the context of the authentic substrate protein and the cell‐based sensor . Therefore, to validate the specificity of BPCS for furin in its cellular context, we first performed site‐directed mutagenesis of the sensor cleavage site.…”

Section: Resultsmentioning

confidence: 99%

“…Functional tests revealed that the sensor recapitulates key features of authentic substrates, including strict SKI-1/S1P specificity and correct subcellular location of processing [31][32][33]. Examination of a panel of SKI-1/S1P sensors with different SKI-1/S1P substrate-derived sequences revealed that the cell-based sensors accurately predicted SKI-1/S1P cleavage efficiency of the authentic substrate [31,32]. In the present study, we used this design as starting point to develop a cell-based basic PC sensor.…”

Section: Introductionmentioning

confidence: 96%

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A novel cell‐based sensor detecting the activity of individual basic proprotein convertases

et al. 2019

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The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides that may not accurately recapitulate the complex cellular context of the basic PC–substrate interaction. Herein we report basic PC sensor (BPCS), a novel cell‐based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. BPCS consists of Gaussia luciferase linked to a sortilin‐1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in the same membrane. Screening of selected candidate peptidomimetic inhibitors revealed that BPCS can readily distinguish between general and selective PC inhibitors in a high‐throughput screening format. The robust and cost‐effective assay format of BPCS makes it suitable to identify novel specific small‐molecule inhibitors against basic PCs for therapeutic application. Its cell‐based nature will allow screening for drug targets in addition to the catalytically active mature enzyme, including maturation, transport, and cellular factors that modulate the enzyme's activity. This broadened ‘target range’ will enhance the likelihood to identify novel small‐molecule compounds that inhibit basic PCs in a direct or indirect manner and represents a conceptual advantage.

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“…S1P is perhaps emerging as a clinical target in diseases such as glioblastoma [4], hypercholesterolemia/cardiovascular disease [5,6], and more prominently as antiviral targets. Pharmacological agents with inhibitory actions on S1P have been shown to reduce dengue virus propagation [7,8], and arenaviral glycoprotein processing [9].…”

Section: Introductionmentioning

confidence: 99%

Membrane-Bound Transcription Factor Site-1 Protease in PF429242 Bound State: Computational Kinetics and Dynamics of Reversible Binding

et al. 2019

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Membrane-bound transcription factor site-1 protease (S1P) is an emerging clinical target due to its roles in lipogenesis, lysosomal biogenesis, unfolded protein response and viral glycoprotein processing. In this study, homology model of S1P was created in order to understand the structural basis for S1P inhibition by PF429242 using molecular docking, molecular dynamics simulation and in silico kinetics studies. PF429242 was docked (GlideScorePF429242=−5.20 kcal/mol) into the catalytic triad (D218, H249 and S414) and validated (R2=0.5686). The reversible binding kinetic parameter (Koff/Kon) was estimated at=7.28E-03 M with fully bound and apo-states interspersed by 3 transient ligand-bound states with unique binding signatures; water plays a major role in PF429242 dissociation from the catalytic site. Communication between key catalytic triad residues is altered in the presence of PF429242. In apo-S1P state, S414–S307/V216-D218 is the preferred route but in PF429242-bound state, S414-S417/V216-D218 is preferred. Communication between S414 and H249 is also shortened in PF429242 bound state; here, only L410 is required unlike apo-state, which requires P418, V256 and F252. Ligand binding did not alter the communication route between S414 and H249 as both recruited D244 and G220. In conclusion, PF429242 binds tightly but reversible to S1P and the details of this interaction has been presented to guide future efforts at developing novel inhibitors. Site-1-protease; PF429242; Kon/Koff; Network analysis

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

Cited by 12 publications

References 42 publications

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

A novel cell‐based sensor detecting the activity of individual basic proprotein convertases

Membrane-Bound Transcription Factor Site-1 Protease in PF429242 Bound State: Computational Kinetics and Dynamics of Reversible Binding

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