Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days of infection. In addition to direct neutralization, in vivo efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal in vivo efficacy afforded by NAbs against SARS-CoV-2

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling

Molecular Design, Functional Characterization and Structural Basis of a Protein Inhibitor Against the HIV-1 Pathogenicity Factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as “wrapping Nef”, is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors

The Phosphofurin Acidic Cluster Sorting Protein 2 (PACS-2) E209K Mutation Responsible for PACS‑2 Syndrome Increases Susceptibility to Apoptosis

Phosphofurin acidic cluster sorting protein 2 (PACS-2) is a multifunctional cytosolic membrane trafficking protein with distinct roles in maintaining cellular homeostasis. Recent clinical reports have described 28 individuals possessing a de novo PACS-2 E209K mutation that present with epileptic seizures and cerebellar dysgenesis. As the PACS-2 E209K missense mutation has become a marker for neurodevelopmental disorders, we sought to characterize its biochemical properties. Accordingly, we observed that the PACS-2 E209K protein exhibited a slower turnover rate relative to PACS-2 wild type (WT) upon cycloheximide treatment in 293T cells. The longer half-life of PACS-2 E209K suggests a disruption in its proteostasis, with the potential for altered protein–protein interactions. Indeed, a regulatory protein in neurodevelopment known as 14-3-3ε was identified as having an increased association with PACS-2 E209K. Subsequently, when comparing the effect of PACS-2 WT and E209K expression on the staurosporine-induced apoptosis response, we found that PACS-2 E209K increased susceptibility to staurosporine-induced apoptosis in HCT 116 cells. Overall, our findings suggest PACS-2 E209K alters PACS-2 proteostasis and favors complex formation with 14-3-3ε, leading to increased cell death in the presence of environmental stressors

Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

AbstractThe immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 (258RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP289). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs

Rapid removal of phagosomal ferroportin in macrophages contributes to nutritional immunity

Abstract Nutrient sequestration is an essential facet of host innate immunity. Macrophages play a critical role in controlling iron availability through expression of the iron transport protein ferroportin (FPN), which extrudes iron from the cytoplasm to the extracellular milieu. During phagocytosis, the limiting phagosomal membrane, which derives from the plasmalemma, can be decorated with FPN and, if functional, will move iron from the cytosol into the phagosome lumen. This serves to feed iron to phagocytosed microbes and would be counterproductive to the many other known host mechanisms working to starve microbes of this essential metal. To understand how FPN is regulated during phagocytosis, we expressed FPN as a green fluorescent protein–fusion protein in macrophages and monitored its localization during uptake of various phagocytic targets, including Staphylococcus aureus, Salmonella enterica serovar Typhimurium, human erythrocytes, and immunoglobulin G opsonized latex beads. We find that FPN is rapidly removed, independently of Vps34 and PI(3)P, from early phagosomes and does not follow recycling pathways that regulate transferrin receptor recycling. Live-cell video microscopy showed that FPN movement on the phagosome is dynamic, with punctate and tubular structures forming before FPN is trafficked back to the plasmalemma. N-ethylmaleimide–sensitive factor, which disrupts soluble NSF attachment protein receptor (SNARE)–mediated membrane fusion and trafficking, prevented FPN removal from the phagosome. Our data support the hypothesis that removal of FPN from the limiting phagosomal membrane will, at the cellular level, ensure that iron cannot be pumped into phagosomes. We propose this as yet another mechanism of host nutritional immunity to subvert microbial growth.</jats:p