Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens

A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection

A community effort in SARS-CoV-2 drug discovery.

peer reviewedThe COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against Covid-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find 'consensus compounds'. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding-, cleavage-, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS-CoV-2 treatments.R-AGR-3826 - COVID19-14715687-CovScreen (01/06/2020 - 31/01/2021) - GLAAB Enric

The Coronavirus E Protein: Assembly and Beyond

The coronavirus E protein is a small membrane protein that has an important role in the assembly of virions. Recent studies have indicated that the E protein has functions during infection beyond assembly, including in virus egress and in the host stress response. Additionally, the E protein has ion channel activity, interacts with host proteins, and may have multiple membrane topologies. The goal of this review is to highlight the properties and functions of the E protein, and speculate on how they may be related

The Hydrophobic Domain of Infectious Bronchitis Virus E Protein Alters the Host Secretory Pathway and Is Important for Release of Infectious Virus ▿

The coronavirus (CoV) E protein plays an important role in virus assembly. The E protein is made in excess during infection and has been shown to have ion channel activity in planar lipid bilayers. However, a role in infection for the unincorporated E or its ion channel activity has not been described. To further investigate the function of the infectious bronchitis virus (IBV) E protein, we developed a recombinant version of IBV in which the E protein was replaced by a mutant containing a heterologous hydrophobic domain. The mutant virus, IBV-EG3, was defective in release of infectious virus particles. Further characterization of IBV-EG3 revealed that damaged particles appeared to accumulate intracellularly. The phenotype of IBV-EG3 suggested that the hydrophobic domain of IBV E may be important for the forward trafficking of cargo, so we determined whether IBV E facilitated the delivery of cargo to the plasma membrane. Surprisingly, we found that IBV E, but not EG3, dramatically reduced the delivery of cargo to the plasma membrane by impeding movement through the Golgi complex. Furthermore, we observed that overexpression of IBV E, but not EG3, induced the disassembly of the Golgi complex. Finally, we determined that the delivery of IBV S to the plasma membrane was reduced in cells infected with wild-type-IBV compared to those infected with IBV-EG3. Our results indicated that the hydrophobic domain of IBV E alters the host secretory pathway to the apparent advantage of the virus

A single polar uncharged residue in IBV E is required for disruption of cargo trafficking.

<p>(A) A helical wheel diagram of the HD of IBV E. Polar uncharged residues are shown in blue; residues mutated to alanine are outlined in red. (B) An immunoblot shows that the alanine mutants of IBV E are expressed and run at a similar molecular weight when transiently expressed in HeLa cells. (C) VSV G was transiently co-expressed with the indicated protein in HeLa cells. 18–22 hours after transfection the cells were pulse-labeled with ³⁵S-methionine/cysteine and chased for 0, 25, and 50 min. VSV G was immunoprecipitated from each sample and digested with endoglycosidase H. The mature (**) and immature (*) forms are indicated. Data from control, IBV E, S13A, and T16 A is shown. (D) Quantification of (C) showing that the T16A mutation inactivates the trafficking block. At each time-point the signal intensity for the mature and immature bands was measured. The percent of endo H resistant VSV G was calculated by dividing the signal for the mature band by the total signal (mature+immature). Data are from at least two independent experiments. Error bars represent +/− SEM.</p

Mutations at T16 in IBV E support VLP production.

<p>(A) Immunoblot showing the amount of IBV N, M, and E in cells and released as VLPs. (10% of cell fraction, 100% of VLP fraction) (B) Quantification of immunoblot data showing the amount of M released with no E, IBV E, T16A, T16S, T16Q or T16N. Data were normalized to the amount of M expressed in each sample, and the amount of M released with IBV E was set to 1 for ease of comparison. Data are from three independent experiments. Error bars represent +/− SEM, and the asterisk denotes a significant decrease in VLP level compared to IBV E by Student's <i>t</i>-test (p<4×10⁻³).</p

T16 is required for Golgi complex disruption.

<p>(A) Indirect immunofluorescence microscopy of HeLa cells transiently expressing IBV E, S13A, or T16A. IBV E is shown in green, GM130 is shown in red and nuclei are shown in blue. (B) Quantification showing the extent to which IBV E and the HD mutants disrupt Golgi complex morphology. To determine the extent of Golgi disruption, the area encompassing GM130 staining was measured in non-transfected cells and in cells expressing the various E mutants as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002674#s4" target="_blank">Materials and Methods</a>. Scale bars, 10 µm. Data are from three independent experiments, N≥54 for each condition. Error bars represent +/− SEM, and the asterisk denotes a significant increase in Golgi disruption compared to the control by Student's <i>t</i>-test (p≤5.4×10⁻³).</p

Generation of IBV E mutants that adopt distinct membrane topologies.

<p>(A) When cells are permeabilized with Triton X-100 both lumen (CFP-KDEL) and cytoplasmic (golgin160-Myc) epitopes are detected. Permeabilization with digitonin allows detection of the cytoplasmic epitope, but not the luminal epitope. (B) Selective permeabilization of cells expressing IBV E, ssIBV E, and FLAG-IBV E. The N-terminus of IBV E and ssIBV E was detected using a rabbit antibody to the N-terminus. The N-terminus of FLAG-IBV E was detected using a mouse anti-FLAG antibody. The C-terminus of each construct was detected using a rat-antibody against the C terminus of IBV E. Scale bars, 10 µm. Cartoons at the bottom of each panel show the predicted topology for each protein. (C) Quantification of topology shown as N-terminus to C-terminus fluorescence ratio (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002674#s4" target="_blank">Material and Methods</a>). The data are normalized to the ratio from the Triton X-100 permeabilized samples. Data are from at least 2 independent experiments with N≥17 for each condition. Error bars represent +/− SEM, and the asterisk denotes a significant decrease in N∶C between the Triton X-100 and digitonin signal by Student's <i>t</i>-test (p≤3.4×10⁻³).</p

Neither ssIBV E or FLAG-IBV E produce normal levels of VLPs.

<p>(A) Immunoblot showing the amount of IBV N, M, and E in cells and released as VLPs. (10% of cell fraction, 100% of VLP fraction) (B) Quantification of immunoblot data showing the amount of M released with no E, IBV E, ssIBVE, or FLAG-IBV E. Data were normalized to the amount of M expressed in each sample, and the amount of M released with IBV E was set to 1 for ease of comparison. Data are from at least five independent experiments. Error bars represent +/− SEM, and the asterisk denotes a significant decrease in VLP level compared to IBV E by Student's <i>t</i>-test (p≤0.01).</p