Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation.

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread

Spatial proteomics defines the content of trafficking vesicles captured by golgin tethers.

Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread

Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C−H Activation by Soybean Lipoxygenase‐1

The ability to relate a hierarchy of protein motions to function remains a compelling experimental challenge at the interface of chemistry and biology. In particular, the proposed contribution of distinctly different classes of local vs. global protein motions during enzymatic catalysis of bond making/breaking processes has been difficult to capture and verify. Herein we employ soybean lipoxygenase-1 as a model system to investigate the impact of high pressure at variable temperatures on the hydrogen tunneling properties of wild type protein and three single site mutants. For all variants, pressure dramatically elevates the experimental enthalpies of activation accompanying the C-H activation step, as predicted for non-physiological conditions that lead to impairment of a protein’s global conformational landscape. In marked contrast, the primary kinetic isotope effects for C-H activation and their corresponding temperature-dependencies remain unchanged up to ca. 700 bar. The differential impact of elevated hydrostatic pressure on the temperature dependencies of rate constants, vs. substrate kinetic isotope effects provides direct experimental verification of two classes of protein motions: local, isotope-dependent donor-acceptor distance sampling modes that are distinct from the more global, isotope independent search for productive protein conformational sub-states

Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C-H Activation by Soybean Lipoxygenase-1.

The proposed contributions of distinct classes of local versus global protein motions during enzymatic bond making/breaking processes has been difficult to verify. We employed soybean lipoxygenase-1 as a model system to investigate the impact of high pressure at variable temperatures on the hydrogen-tunneling properties of the wild-type protein and three single-site mutants. For all variants, pressure dramatically elevates the enthalpies of activation for the C-H activation. In contrast, the primary kinetic isotope effects (KIEs) for C-H activation and their corresponding temperature dependencies remain unchanged up to ca. 700 bar. The differential impact of elevated hydrostatic pressure on the temperature dependencies of rate constants versus substrate KIEs provides direct evidence for two distinct classes of protein motions: local, isotope-dependent donor-acceptor distance-sampling modes, and a more global, isotope-independent search for productive protein conformational sub-states

Spatial proteomics defines the content of trafficking vesicles captured by golgin tethers.

Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

<div><p>The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in <i>Caenorhabditis elegans</i> for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.</p></div

EIPR-1 interacts with members of the GARP and EARP complexes.

<p>(A) The rat ortholog of EIPR-1 is expressed in 832/13 cells. Left and middle: RT-PCR shows that rat orthologs of CCCP-1, RAB-2, EIPR-1 and RUND-1 are expressed in 832/13 cells. All bands are of the predicted size for the full-length cDNA except for CCCP1, which is truncated and corresponds to the N-terminal half of the gene. (B) EIPR1 interacts with members of the GARP and EARP complexes. List of top hits from mass spectrometry of a pulldown of rEIPR1::GFP in 832/13 cells after subtracting hits found in GFP control pulldowns. # seq = number of unique peptides from each protein. All proteins with more than 5 unique peptides are shown from one of two independent experiments. More complete data tables for both experiments are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006074#pgen.1006074.s005" target="_blank">S5A Fig</a>. (C) EIPR1 interacts with VPS51 and VPS50. EGFP-tagged rat EIPR1 or EGFP was coexpressed with mCherry-tagged rat VPS51 in 832/13 cells. Immunoprecipitation of EIPR1::EGFP pulled down VPS51::mCherry and endogenous VPS50. Immunoprecipitation of untagged EGFP did not pull down VPS51::mCherry or VPS50. IN: input; IP: immunoprecipitation.</p

<i>eipr-1</i> mutants have defects in trafficking dense-core vesicle cargos.

<p>(A) Left: representative images of NLP-21::Venus (<i>nuIs183</i> transgene) fluorescence in motor neuron axons of the dorsal nerve cord. Scale bar: 10 μm. Right: quantification of NLP-21::Venus fluorescence levels in the dorsal nerve cord. The mean fluorescence intensity is given in arbitrary units. <i>eipr-1(tm4790)</i>, <i>rab-2(nu415)</i>, and <i>rund-1(tm3622)</i> mutants have decreased levels of fluorescence in the dorsal cord (DC), indicative of an NLP-21::Venus sorting or trafficking defect (***, P<0.001 compared to wild type). Double mutants of <i>eipr-1(tm4790)</i> with <i>rab-2(nu415)</i> or <i>rund-1(tm3622)</i> are not significantly different than the single mutants (P>0.05). Error bars = SEM; n = 10. (B) FLP-3::Venus (<i>ceIs61</i> transgene) fluorescence levels in the dorsal nerve cord. ***, P<0.001. Error bars = SEM; n = 12. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006074#pgen.1006074.s003" target="_blank">S3A Fig</a> for representative images. (C) INS-22::Venus <i>(nuIs195</i> transgene) fluorescence levels in the dorsal nerve cord. ***, P<0.001. Error bars = SEM; n = 11–12. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006074#pgen.1006074.s003" target="_blank">S3B Fig</a> for representative images. (D) IDA-1::GFP (<i>ceIs72</i> transgene) fluorescence levels in the dorsal nerve cord. **, P<0.01. Error bars = SEM; n = 13–14. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006074#pgen.1006074.s003" target="_blank">S3C Fig</a> for representative images. (E) UNC-17::GFP (<i>oxSi702</i> transgene) fluorescence levels in the dorsal nerve cord. <i>eipr-1</i> mutants do not have a defect (P>0.05). Scale bar: 10 μm. Error bars = SEM; n = 16–19. (F) The peptide processing mutant <i>egl-3(ok979)</i> increases the level of NLP-21::Venus (<i>nuIs183</i> transgene) in the dorsal cord in both wild type and <i>eipr-1(tm4790)</i> mutant backgrounds. Left: representative images. Scale bar: 10 μm. Right: quantification of NLP-21::Venus fluorescence levels in the dorsal nerve cord. ***, P<0.001. Error bars = SEM; n = 9–12.</p