Hall, B. S. | Search bioRxiv

bioRxiv2021

Mycolactone enhances the Ca2+ leakage from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of, the major translocon subunit Sec61, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61 because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61 in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon "breathing" during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactones stabilisation of the latter underpins the phenotype observed.

biochemistry↗

bioRxiv2021

Murine Norovirus infection results in anti-inflammatory response downstream of amino acids depletion in macrophages

Murine norovirus (MNV) infection results in a late translation shut-off, that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the eIF2 kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signalling during metabolic stress. While viral infection is usually associated with activation of dsRNA binding pattern recognition receptor PKR, we hypothesised that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterise cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signalling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. ImportanceDuring viral infection, host defences are typically characterised by the secretion of pro-inflammatory autocrine and paracrine cytokines, potentiation of the IFN response and induction of the anti-viral response via activation of JAK and Stat signalling. To avoid these and propagate viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signalling. This highlights novel tools in the viral countermeasures tool-kit, and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

microbiology↗