Modulation of in Vitro SARS-CoV-2 Infection by Stephania tetrandra and Its Alkaloid Constituents.

Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography-mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.

General strategies for using amino acid sequence data to guide biochemical investigation of protein function.

The rapid increase of '-omics' data warrants the reconsideration of experimental strategies to investigate general protein function. Studying individual members of a protein family is likely insufficient to provide a complete mechanistic understanding of family functions, especially for diverse families with thousands of known members. Strategies that exploit large amounts of available amino acid sequence data can inspire and guide biochemical experiments, generating broadly applicable insights into a given family. Here we review several methods that utilize abundant sequence data to focus experimental efforts and identify features truly representative of a protein family or domain. First, coevolutionary relationships between residues within primary sequences can be successfully exploited to identify structurally and/or functionally important positions for experimental investigation. Second, functionally important variable residue positions typically occupy a limited sequence space, a property useful for guiding biochemical characterization of the effects of the most physiologically and evolutionarily relevant amino acids. Third, amino acid sequence variation within domains shared between different protein families can be used to sort a particular domain into multiple subtypes, inspiring further experimental designs. Although generally applicable to any kind of protein domain because they depend solely on amino acid sequences, the second and third approaches are reviewed in detail because they appear to have been used infrequently and offer immediate opportunities for new advances. Finally, we speculate that future technologies capable of analyzing and manipulating conserved and variable aspects of the three-dimensional structures of a protein family could lead to broad insights not attainable by current methods.

Azorhizobium caulinodans Chemotaxis Is Controlled by an Unusual Phosphorelay Network.

Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms root nodules on its host legume, Sesbania rostrata. This agriculturally significant symbiotic relationship is important in lowland rice cultivation and allows nitrogen fixation under flood conditions. Chemotaxis plays an important role in bacterial colonization of the rhizosphere. Plant roots release chemical compounds that are sensed by bacteria, triggering chemotaxis along a concentration gradient toward the roots. This gives motile bacteria a significant competitive advantage during root surface colonization. Although plant-associated bacterial genomes often encode multiple chemotaxis systems, A. caulinodans appears to encode only one. The che cluster on the A. caulinodans genome contains cheA, cheW, cheY2, cheB, and cheR. Two other chemotaxis genes, cheY1 and cheZ, are located independently from the che operon. Both CheY1 and CheY2 are involved in chemotaxis, with CheY1 being the predominant signaling protein. A. caulinodans CheA contains an unusual set of C-terminal domains: a CheW-like/receiver pair (termed W2-Rec) follows the more common single CheW-like domain. W2-Rec impacts both chemotaxis and CheA function. We found a preference for transfer of phosphoryl groups from CheA to CheY2, rather than to W2-Rec or CheY1, which appears to be involved in flagellar motor binding. Furthermore, we observed increased phosphoryl group stabilities on CheY1 compared to CheY2 and W2-Rec. Finally, CheZ enhanced dephosphorylation of CheY2 substantially more than CheY1 but had no effect on the dephosphorylation rate of W2-Rec. This network of phosphotransfer reactions highlights a previously uncharacterized scheme for regulation of chemotactic responses. IMPORTANCE Chemotaxis allows bacteria to move toward nutrients and away from toxins in their environment. Chemotactic movement provides a competitive advantage over nonspecific motion. CheY is an essential mediator of the chemotactic response, with phosphorylated and unphosphorylated forms of CheY differentially interacting with the flagellar motor to change swimming behavior. Previously established schemes of CheY dephosphorylation include action of a phosphatase and/or transfer of the phosphoryl group to another receiver domain that acts as a sink. Here, we propose that A. caulinodans uses a concerted mechanism in which the Hpt domain of CheA, CheY2, and CheZ function together as a dual sink system to rapidly reset chemotactic signaling. To the best of our knowledge, this mechanism is unlike any that have previously been evaluated. Chemotaxis systems that utilize both receiver and Hpt domains as phosphate sinks likely occur in other bacterial species.

Pseudomonas Virulence Factor Pathway Synthesizes Autoinducers That Regulate the Secretome of a Pathogen.

Cell-to-cell communication via chemical signals is an essential mechanism that pathogenic bacteria use to coordinate group behaviors and promote virulence. The Pseudomonas virulence factor (pvf) gene cluster is distributed in more than 500 strains of proteobacteria including both plant and human pathogens. The pvf cluster has been implicated in the production of signaling molecules important for virulence; however, the regulatory impact of these signaling molecules on virulence had not been elucidated. Using the insect pathogen Pseudomonas entomophila L48 as a model, we demonstrated that pvf-encoded biosynthetic enzymes produce PVF autoinducers that regulate the expression of pvf genes and a gene encoding the toxin monalysin via quorum sensing. In addition, PVF autoinducers regulate the expression of nearly 200 secreted and membrane proteins, including toxins, motility proteins, and components of the type VI secretion system, which play key roles in bacterial virulence, colonization, and competition with other microbes. Deletion of pvf also altered the secondary metabolome. Six major compounds upregulated by PVF autoinducers were isolated and structurally characterized, including three insecticidal 3-indolyl oxazoles, the labradorins, and three antimicrobial pyrrolizidine alkaloids, the pyreudiones. The signaling properties of PVF autoinducers and their wide-ranging regulatory effects indicate multifaceted roles of PVF in controlling cell physiology and promoting virulence. The broad genome distribution of pvf suggests that PVF-mediated signaling is relevant to many bacteria of agricultural and biomedical significance.

An Epstein-Barr Virus MicroRNA Blocks Interleukin-1 (IL-1) Signaling by Targeting IL-1 Receptor 1.

Epstein-Barr virus (EBV) encodes >44 viral microRNAs (miRNAs) that are differentially expressed throughout infection, can be detected in Epstein-Barr virus (EBV)-positive tumors, and manipulate several biological processes, including cell proliferation, apoptosis, and immune responses. Here, we show that EBV BHRF1-2 miRNAs block NF-κB activation following treatment with proinflammatory cytokines, specifically interleukin-1ß (IL-1ß). Analysis of EBV PAR-CLIP miRNA targetome data sets combined with pathway analysis revealed multiple BHRF1-2 miRNA targets involved in interleukin signaling pathways. By further analyzing changes in cellular gene expression patterns, we identified the IL-1 receptor 1 (IL1R1) as a direct target of miR-BHRF1-2-5p. Targeting the IL1R1 3' untranslated region (UTR) by EBV miR-BHRF1-2-5p was confirmed using 3'-UTR luciferase reporter assays and Western blot assays. Manipulation of EBV BHRF1-2 miRNA activity in latently infected B cells altered steady-state cytokine levels and disrupted IL-1ß responsiveness. These studies demonstrate functionally relevant BHRF1-2 miRNA interactions during EBV infection, which is an important step in understanding their roles in pathogenesis.IMPORTANCE IL-1 signaling plays an important role in inflammation and early activation of host innate immune responses following virus infection. Here, we demonstrate that a viral miRNA downregulates the IL-1 receptor 1 during EBV infection, which consequently alters the responsiveness of cells to IL-1 stimuli and changes the cytokine expression levels within infected cell populations. We postulate that this viral miRNA activity not only disrupts IL-1 autocrine and paracrine signaling loops that can alert effector cells to sites of infection but also provides a survival advantage by dampening excessive inflammation that may be detrimental to the infected cell.

Japanese Macaque Rhadinovirus Encodes a Viral MicroRNA Mimic of the miR-17 Family.

UNLABELLED: Japanese macaque (JM) rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from JM with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immunohistopathology comparable to those of multiple sclerosis in humans. Little is known about the molecular biology of JMRV. Here, we sought to identify and characterize the small RNAs expressed during lytic JMRV infection using deep sequencing. Fifteen novel viral microRNAs (miRNAs) were identified in JMRV-infected fibroblasts, all of which were readily detectable by 24 h postinfection and accumulated to high levels by 72 h. Sequence comparisons to human Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs revealed several viral miRNA homologs. To functionally characterize JMRV miRNAs, we screened for their effects on nuclear factor kappa B (NF-κB) signaling in the presence of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). Multiple JMRV miRNAs suppressed cytokine-induced NF-κB activation. One of these miRNAs, miR-J8, has seed sequence homology to members of the cellular miR-17/20/106 and miR-373 families, which are key players in cell cycle regulation as well as inflammation. Using reporters, we show that miR-J8 can target 3' untranslated regions (UTRs) with miR-17-5p or miR-20a cognate sites. Our studies implicate JMRV miRNAs in the suppression of innate antiviral immune responses, which is an emerging feature of many viral miRNAs. IMPORTANCE: Gammaherpesviruses are associated with multiple diseases linked to immunosuppression and inflammation, including AIDS-related cancers and autoimmune diseases. JMRV is a recently identified herpesvirus that has been linked to JME, an inflammatory demyelinating disease in Japanese macaques that mimics multiple sclerosis. There are few large-animal models for gammaherpesvirus-associated pathogenesis. Here, we provide the first experimental evidence of JMRV miRNAs in vitro and demonstrate that one of these viral miRNAs can mimic the activity of the cellular miR-17/20/106 family. Our work provides unique insight into the roles of viral miRNAs during rhadinovirus infection and provides an important step toward understanding viral miRNA function in a nonhuman primate model system.