Ebolaviruses are emerging pathogens that cause severe and often fatal viral hemorrhagic fevers. Four distinct ebolaviruses are known to cause Ebola virus disease in humans. The ebolavirus envelope glycoprotein (GP) is heavily glycosylated, but the precise glycosylation patterns of ebolaviruses are largely unknown. Here we demonstrate that approximately 50 different N-glycan structures are present in GP derived from the four pathogenic ebolaviruses, including high mannose, hybrid, and bi-, tri-, and tetra-antennary complex glycans with and without fucose and sialic acid. The overall N-glycan composition is similar between the different ebolavirus GPs. In contrast, the amount and type of O-glycan structures varies widely between ebolavirus GPs. Notably, this O-glycan dissimilarity is also present between two variants of Ebola virus, the original Yambuku variant and the Makona variant responsible for the most recent Western African epidemic. The data presented here should serve as the foundation for future ebolaviral entry and immunogenicity studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress occurs by lysosomal exocytosis. We show that the Spike D614G mutation enhances Spike trafficking to lysosomes, drives Spike-mediated reprogramming of lysosomes, and reduces cell surface Spike expression by ~3-fold. D614G is not a human-specific adaptation. Rather, it is an adaptation to the earlier furin cleavage site insertion (FCSI) mutation that occurred at the genesis of SARS-CoV-2. While advantageous to the virus, furin cleavage of spike has deleterious effects on spike structure and function, inhibiting its trafficking to lysosomes and impairing its infectivity by the transmembrane serine protease 2(TMPRSS2)-independent, endolysosomal pathway. D614G restores spike trafficking to lysosomes and enhances the earliest events in SARS-CoV-2 infectivity, while spike mutations that restore SARS-CoV-2’s TMPRSS2-independent infectivity restore spike’s trafficking to lysosomes. Together, these and other results show that D614G is an intragenic suppressor of deleterious traits linked to the FCSI and lend additional support to the endolysosomal model of SARS-CoV-2 egress and entry.

Candidemia is the most common bloodstream infection in the United States. During 23 infection, the fungal cell wall is an important virulence factor, playing roles in adhesion, 24 immune recognition and colonization. The human innate immune system recognizes β-25 glucan, a highly immunogenic component of the fungal cell wall. During innate immune 26 recognition of Candida, the organization of cell wall β-glucan is an important 27 determinant of a successful immune activation. However, there have been many reports 28 showing conflicting biological activities of β-glucans with different size, branching and 29 structure. Here, using quantitative fluorescence imaging techniques, we investigate how 30 differential size and structure of β-glucan impacts activation of the innate immune 31 receptor, Dectin-1A. Our results indicate a positive correlation between highly structured 32 glucans and Dectin-1A activation. Furthermore, we determined this is due to the higher 33 ordered β-glucan causing Dectin-1A receptors to form aggregates that are below 15 nm 34 in size. Finally, Dectin-1A receptor aggregation has also been shown to form at fungal 35 particle contact sites with high β-glucan exposure. 36 Abstract 37Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3:1,6)-glucan, 38 a structural component of Candida species cell walls. The higher order structure of β-39 glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary 40 (helical) and quaternary structure. Model Saccharomyces cerevisiae β-glucans of 41 medium and high molecular weight (MMW and HMW, respectively) are highly 42 structured. In contrast, low MW glucan (LMW) is much less structured. Despite similar 43 affinity for Dectin-1A, the ability of glucans to induce Dectin-1A mediated calcium influx 3 44and Syk phosphorylation positively correlates with their degree of higher order structure. 45Chemical denaturation and renaturation of MMW glucan showed that glucan structure 46 determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the 47 6 113 ITAM domains poorly recruit and activate Syk for downstream signaling [44], suggesting 114 that hemITAM domains with their single phosphotyrosine site would be poorly activating 115 in a monomeric configuration. Consistent with this hypothesis, another (hem)ITAM 116 bearing receptor, CLEC-2, is reported to require dimerization for its signaling [45]. By 117 analogy to this and other (hem)ITAM receptors, it is hypothesized that Dectin-1A must 118 oligomerize to recapitulate a multivalent binding site for Syk and to facilitate signal 119 transduction [8,45,46]. However, this prediction has not been directly explored for 120Dectin-1A in live cells at the molecular level with relation to structural determinants of 121 receptor-ligand complex organization and signaling outcomes. 122In this study, we propose that factors that induce an aggregated membrane organization 123 of Dectin-1A during activation are very important for determining signaling ...

Filoviruses are highly virulent pathogens capable of causing severe disease. The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding. As such, they are the main candidate vaccine antigen. Despite their virulence, most filoviruses are not comprehensively characterized, and relatively few commercially produced reagents are available for their study. Here, we describe two methods for production and purification of filovirus glycoproteins in insect and mammalian cell lines. Considerations of expression vector choice, modifications to sequence, troubleshooting of purification method, and glycosylation differences are all important for successful expression of filovirus glycoproteins in cell lines. Given the scarcity of commercially available filovirus glycoproteins, we hope our experiences with possible difficulties in purification of the proteins will facilitate other researchers to produce and purify filovirus glycoproteins rapidly.

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