Inhibitors of the small membrane (M) protein viroporin prevent Zika virus infection
- University of Leeds, United Kingdom
- MRC University of Glasgow Centre for Virus Research, United Kingdom
Abstract
Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small Membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics (MD) simulations. Models contained a predicted lumenal rimantadine binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted-therapies.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files; access to MD data and molecular models may be requested and, if accepted, accessed via MTA. Raw simulation data can be accessed via the Leeds Data Repository (https://archive.researchdata.leeds.ac.uk/) at the following DOI: https://doi.org/10.5518/1505
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ZIKV M protein simulation dataLeeds Data Repository, 974.
Article and author information
Author details
Daniella A Lefteri
Funding
Medical Research Council (G0700124)
- Matthew J Bentham
- Stephen Griffin
University of Leeds (LIMR Studentship)
- Emma Brown
- Richard Foster
- Clive S McKimmie
- Antreas C Kalli
- Stephen Griffin
Medical Research Council (MC_UU_12014/8)
- Claire Donald
- Alain Kohl
Medical Research Council (MR/N017552/1)
- Claire Donald
- Alain Kohl
University of Leeds (LIMR Studentship)
- Daniella A Lefteri
- Clive S McKimmie
- Stephen Griffin
Medical Research Council (MR/T016205/1)
- Amy Moran
- Stephen Griffin
UK Research and Innovation (Impact Acceleration Account ( IAA))
- Gemma Swinscoe
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Procedures were carried out in accordance with the United Kingdom Home Office regulations under the authority of the appropriate project and personal license (awarded to CSM, and CSM/DL respectively).
Copyright
© 2024, Brown et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Inhibitors of the small membrane (M) protein viroporin prevent Zika virus infection
eLife 13:e68404.
https://doi.org/10.7554/eLife.68404
Further reading
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
