Human rhinovirus promotes STING trafficking to replication organelles to promote viral replication

Human rhinovirus (HRV), like coronavirus (HCoV), are positive-strand RNA viruses that cause both upper and lower respiratory tract illness, with their replication facilitated by concentrating RNA-synthesizing machinery in intracellular compartments made of modified host membranes, referred to as replication organelles (ROs). Here we report a non-canonical, essential function for stimulator of interferon genes (STING) during HRV infections. While the canonical function of STING is to detect cytosolic DNA and activate inflammatory responses, HRV infection triggers the release of STIM1-bound STING in the ER by lowering Ca2+, thereby allowing STING to interact with phosphatidylinositol 4-phosphate (PI4P) and traffic to ROs to facilitates viral replication and transmission via autophagy. Our results thus hint a critical function of STING in HRV viral replication and transmission, with possible implications for other RO-mediated RNA viruses

Prediction of Drought-Resistant Genes in Arabidopsis thaliana Using SVM-RFE

Background: Identifying genes with essential roles in resisting environmental stress rates high in agronomic importance. Although massive DNA microarray gene expression data have been generated for plants, current computational approaches underutilize these data for studying genotype-trait relationships. Some advanced gene identification methods have been explored for human diseases, but typically these methods have not been converted into publicly available software tools and cannot be applied to plants for identifying genes with agronomic traits. Methodology: In this study, we used 22 sets of Arabidopsis thaliana gene expression data from GEO to predict the key genes involved in water tolerance. We applied an SVM-RFE (Support Vector Machine-Recursive Feature Elimination) feature selection method for the prediction. To address small sample sizes, we developed a modified approach for SVM-RFE by using bootstrapping and leave-one-out cross-validation. We also expanded our study to predict genes involved in water susceptibility. Conclusions: We analyzed the top 10 genes predicted to be involved in water tolerance. Seven of them are connected to known biological processes in drought resistance. We also analyzed the top 100 genes in terms of their biological functions. Our study shows that the SVM-RFE method is a highly promising method in analyzing plant microarray data for studyin

Synthetic and structural investigations of the novel biologically active cyclodepsipeptides: The didemnins

The didemnins, isolated from a marine tunicate displayed diverse bioactivity namely antiviral, antitumor, and immunosuppressive. Didemnin B, one of the most successful drug candidate of the Didemnidae family has been the target of our investigations. Most didemnins contain a common macrocycle and differ only in the side chains attached to the amino group of threonine (Figure 1.2). Didemnin B has greater immunosuppressive activity than cyclosporin A. Phase II clinical trials for antitumor activity showed no significant activity yet demonstrated toxicity possibly due to its rapid conversion to a toxic metabolite. In recent years, peptidomimetics have offered great potential to produce compounds with enhanced enzymatic stability and reduced toxicity. In order to arrive at the pharmacophore when little is known regarding the receptor-bound conformation, structure-activity relationships are extensively used. Our preliminary SAR investigations are aimed at confirming the original hypothesis that $\beta$-turn side chain of the didemnin B is the main if not the only bioactive site and the macrocycle is used only for binding or anchoring to the receptor . The syntheses and biological activities of the $\beta$-turn analogs (14-16, Figure 1.6) that were designed, retaining the residues found in the $\beta$-turn region of the natural compound, are presented in chapter 1. Also shown in chapter 1, is the syntheses of acyclic analogs of didemnins in order to investigate effects of ring-opening on bioactivity. Introduction of covalent linkers to replace amino acids that are not required for biological activity have produced active analogs. The reduced ring analog (23, Figure 2.1), that we have designed with the aid of molecular modeling, displayed remarkable superimposition with the natural compound. The syntheses of this analog as well as the syntheses of two other analogs are presented in chapter 2. In chapter 3, a bolder approach was taken that is to replace the entire peptide backbone with a suitable scaffolding. We chose 2,3-diaminoglycal as a scaffolding that would keep the pharmacaphores in a presumed bioactive conformation. Progress made toward the synthesis of this analog (89, Figure 3.1) is discussed in chapter 3

Synthetic and structural investigations of the novel biologically active cyclodepsipeptides: The didemnins

The didemnins, isolated from a marine tunicate displayed diverse bioactivity namely antiviral, antitumor, and immunosuppressive. Didemnin B, one of the most successful drug candidate of the Didemnidae family has been the target of our investigations. Most didemnins contain a common macrocycle and differ only in the side chains attached to the amino group of threonine (Figure 1.2). Didemnin B has greater immunosuppressive activity than cyclosporin A. Phase II clinical trials for antitumor activity showed no significant activity yet demonstrated toxicity possibly due to its rapid conversion to a toxic metabolite. In recent years, peptidomimetics have offered great potential to produce compounds with enhanced enzymatic stability and reduced toxicity. In order to arrive at the pharmacophore when little is known regarding the receptor-bound conformation, structure-activity relationships are extensively used. Our preliminary SAR investigations are aimed at confirming the original hypothesis that $\beta$-turn side chain of the didemnin B is the main if not the only bioactive site and the macrocycle is used only for binding or anchoring to the receptor . The syntheses and biological activities of the $\beta$-turn analogs (14-16, Figure 1.6) that were designed, retaining the residues found in the $\beta$-turn region of the natural compound, are presented in chapter 1. Also shown in chapter 1, is the syntheses of acyclic analogs of didemnins in order to investigate effects of ring-opening on bioactivity. Introduction of covalent linkers to replace amino acids that are not required for biological activity have produced active analogs. The reduced ring analog (23, Figure 2.1), that we have designed with the aid of molecular modeling, displayed remarkable superimposition with the natural compound. The syntheses of this analog as well as the syntheses of two other analogs are presented in chapter 2. In chapter 3, a bolder approach was taken that is to replace the entire peptide backbone with a suitable scaffolding. We chose 2,3-diaminoglycal as a scaffolding that would keep the pharmacaphores in a presumed bioactive conformation. Progress made toward the synthesis of this analog (89, Figure 3.1) is discussed in chapter 3

A diamidobenzimidazole STING agonist protects against SARS-CoV-2 infection

Coronaviruses are a family of RNA viruses that cause acute and chronic diseases of the upper and lower respiratory tract in humans and other animals. SARS-CoV-2 is a recently emerged coronavirus that has led to a global pandemic causing a severe respiratory disease known as COVID-19 with significant morbidity and mortality worldwide. The development of antiviral therapeutics are urgently needed while vaccine programs roll out worldwide. Here we describe a diamidobenzimidazole compound, diABZI-4, that activates STING and is highly effective in limiting SARS-CoV-2 replication in cells and animals. diABZI-4 inhibited SARS-CoV-2 replication in lung epithelial cells. Administration of diABZI-4 intranasally before or even after virus infection conferred complete protection from severe respiratory disease in K18-ACE2-transgenic mice infected with SARS-CoV-2. Intranasal delivery of diABZI-4 induced a rapid short-lived activation of STING, leading to transient proinflammatory cytokine production and lymphocyte activation in the lung associated with inhibition of viral replication. Our study supports the use of diABZI-4 as a host-directed therapy which mobilizes antiviral defenses for the treatment and prevention of COVID-19

MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

<div><p>CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4⁺ and CD8⁺ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. <i>In vivo</i>, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses <i>in vivo</i>.</p></div

Mixed inflammatory infiltrates of T cells, B cells, and macrophages in MALT1 protease-dead mice.

<p>IHC photomicrographs (80X magnification, insets 400X magnification) of inflammatory cell infiltrates surrounding vessels (V) of the lung and tunica adventicia of glandular stomach in <i>Malt1</i>^PD/PD mice, using antibodies against: (A, D) CD3 to detect T cells; (B, E) CD45 to detect B-cell; and (C, F) F4/80 to detect macrophage. Non-selective background staining was higher with the F4/80 antibody, as evident with the pale staining of erythrocytes within the vessel lumen (V, Fig 8C). Therefore, macrophage identification was made by considering the higher staining intensity and morphology of cells stained (inset, Fig 8C). IHC photomicrographs of inflammatory cell infiltrates in lung of <i>Malt1</i>^PD/PD mice (8A, C) demonstrates a mixed population of T-cells, B-cells and macrophages comprising the perivascular cuffs of pulmonary vessels. Similar to the lung infiltrates of <i>Malt1</i>^PD/PD, inflammatory infiltrates in deeper glandular portions of stomach had mixed population of T cells, B cells and macrophages in the tunica adventicia (Fig 8D, F).</p

MALT1 protease activity is required for maximal T-dependent and T-independent antibody responses <i>in vivo</i>.

<p>(A) <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice (n = 6/group) were immunized with the T-dependent antigen KLH, and serum anti-KLH antibody titers were measured by ELISA on days 7, 14, 21 and 28 after immunization. (B) <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice (n = 4-5/group) were immunized with the T-independent antigen TNP-Ficoll, and serum anti-TNP antibody titers were measured by ELISA on days 7, 14, 21 and 28 after immunization. Data represents Ig levels in individual animals, and averages are indicated by horizontal lines. Significance was determined relative to the <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01, †<i>p</i> < 0.001.</p

CLR stimulation of cytokine production by BMDCs involves MALT1 protease activity.

<p>BMDCs from <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice were stimulated with (A) curdlan to activate Dectin-1, (B) anti-Dectin-2 antibody to activate Dectin-2, and (C) TDB to activate Mincle. IFN-γ, IL-1β, IL-10, IL-12 p70, IL-6, KC (IL-8 homologue), and TNF-α levels were measured after 20 h by MSD, and results are shown only for cytokines for which significant expression was detected. Maximum cytokine concentration ranges detected were: curdlan, 1–3 ng/ml IL-1β, 4–20 ng/ml IL-6, 4–9 ng/ml KC, 0.5–1 ng/ml IL-10, 0.2–0.4 ng/ml IL-12p70, and 40–100 ng/ml TNF-α; anti-Dectin-2, 0.1–0.3 ng/ml IL-6, 0.5–2 ng/ml KC, and 3–15 ng/ml TNF-α; TDB, 0.3–0.6 ng/ml KC, and 0.3–2 ng/ml TNF-α. Where indicated, cells were treated 126 μM Z-VRPR-FMK (VRPR) for 45 min prior to stimulation. All bar graphs represent the average response expressed as a percent of the stimulated <i>Wt</i> control for 6 mice/group, and are representative of 3 or more studies. Significance was determined relative to the stimulated <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01, †<i>p</i> < 0.001, ‡<i>p</i> < 0.0001. Error bars represent +/- SEM.</p

A C472A mutation in MALT1 inactivates protease activity without changing protein expression.

<p>(A) Homologous recombination of the <i>Wt Malt1</i> locus (<i>I</i>) in C57BL/6-derived embryonic stem cells was used to introduce a catalytically inactive C472A mutation into exon 12 of the <i>Malt1</i> gene (for clarity, the genomic locus is not draw to scale). The resulting neo cassette-containing mice (<i>II</i>) were crossed to a Flp recombinase-expressing deleter mouse strain to generate animals carrying the protease-dead allele (<i>III</i>) used in these studies. These mice were in turn crossed to a Cre recombinase-expressing deleter strain to excise exon 12 and generate a null allele (<i>IV</i>). Close triangles, FRT sites; open triangles, <i>loxP</i> sites. (B) Purified total B cells from the spleens of <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice were treated with (+) or without (-) PMA plus ionomycin for 1 h and assessed by Western blotting for expression of MALT1, CYLD, Bcl10, and proteolytically cleaved CYLD and Bcl10. For clarity, the images were cropped. Uncropped western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127083#pone.0127083.s001" target="_blank">S1 Fig</a> (Complete western blots confirming MALT1 (C472A) expression and loss of protease activity).</p