Inter-compatibility of eukaryotic and Asgard archaea ribosome-translocon machineries.

In all domains of life, the ribosome-translocon complex inserts nascent transmembrane proteins into, and processes and transports signal peptide-containing proteins across, membranes. Eukaryotic translocons are anchored in the endoplasmic reticulum, while the prokaryotic complexes reside in cell membranes. Phylogenetic analyses indicate inheritance of eukaryotic Sec61/OST/TRAP translocon subunits from an Asgard archaea ancestor. However, the mechanism for translocon migration from a peripheral membrane to an internal cellular compartment (the proto-endoplasmic reticulum) during eukaryogenesis is unknown. Here we show compatibility between the eukaryotic ribosome-translocon complex and Asgard signal peptides and transmembrane proteins. We find that Asgard translocon proteins from Candidatus Prometheoarchaeum syntrophicum strain MK-D1, a Lokiarchaeon confirmed to contain no internal cellular membranes, are targeted to the eukaryotic endoplasmic reticulum on ectopic expression. Furthermore, we show that the cytoplasmic domain of MK-D1 OST1 (ribophorin I) can interact with eukaryotic ribosomes. Our data indicate that the location of existing ribosome-translocon complexes, at the protein level, determines the future placement of yet to be translated translocon subunits. This principle predicts that during eukaryogenesis, under positive selection pressure, the relocation of a few translocon complexes to the proto-endoplasmic reticulum will have contributed to propagating the new translocon location, leading to their loss from the cell membrane.

Attenuation of N-glycosylation causes polarity and adhesion defects in the C. elegans embryo.

The Caenorhabditiselegans early embryo is highly polarized, requiring sequestration of cytoplasmic polarity factors at the plasma membrane. This compartmentalization aids asymmetric distribution of lipids and proteins, which is partially responsible for the fates of the daughter cells. Since most plasma membrane proteins are glycosylated, we determined the effect of attenuation of N-glycosylation on cell polarity. While polarity establishment was not perturbed, the size difference between the two cells formed in first cell division (AB and P1) was more variable in embryos with reduced N-glycosylation than in the mock-treated embryos. In addition, among other deficiencies, we observed spindle orientation defects in two-cell embryos. Moreover, cell-cell adhesion was specifically lost at the two-cell stage when N-glycosylation was reduced. This loss-of-adhesion phenotype was rescued by interfering with polarity establishment, indicating that polarity establishment enforces plasma membrane compartmentalization. Consistent with this idea, the decreased plasma membrane levels of the adhesion proteins E-cadherin and MAGI-1 in ribo-1(RNAi) embryos were restored in the absence of functional PAR-2. Our data suggest a general role for N-glycosylation in plasma membrane compartmentalization and cell polarity.

Interferon-Inducible Guanylate-Binding Protein 5 Inhibits Replication of Multiple Viruses by Binding to the Oligosaccharyltransferase Complex and Inhibiting Glycoprotein Maturation.

Viral infection induces production of type I interferons and expression of interferon-stimulated genes (ISGs) that play key roles in inhibiting viral infection. Here, we show that the ISG guanylate-binding protein 5 (GBP5) inhibits N-linked glycosylation of key proteins in multiple viruses, including SARS-CoV-2 spike protein. GBP5 binds to accessory subunits of the host oligosaccharyltransferase (OST) complex and blocks its interaction with the spike protein, which results in misfolding and retention of spike protein in the endoplasmic reticulum likely due to decreased N-glycan transfer, and reduces the assembly and release of infectious virions. Consistent with these observations, pharmacological inhibition of the OST complex with NGI-1 potently inhibits glycosylation of other viral proteins, including MERS-CoV spike protein, HIV-1 gp160, and IAV hemagglutinin, and prevents the production of infectious virions. Our results identify a novel strategy by which ISGs restrict virus infection and provide a rationale for targeting glycosylation as a broad antiviral therapeutic strategy.

The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis.

Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus - a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered, stabilizing association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling.

EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.