Class I benzoyl-coenzyme A (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of the central BzCoA intermediate and analogues of it to conjugated cyclic 1,5-dienoyl-CoAs probably by a radical-based, Birch-like reduction mechanism. Discovered in 1995, the enzyme from the denitrifying bacterium Thauera aromatica (BCRTar) has so far remained the only isolated and biochemically accessible BCR, mainly because BCRs are extremely labile and their heterologous production has largely failed, so far. Here, we describe a platform for the heterologous expression of the four structural genes encoding a designated 3-methylbenzoylCoA reductase from the related denitrifying species Thauera chlorobenzoica (MBRTcl) in Escherichia coli This reductase represents the prototype of a distinct subclass of ATP-dependent BCRs that were proposed to be involved in the degradation of methyl-substituted BzCoA analogues. The recombinant MBRTcl had an αβγδ-subunit architecture, contained three low-potential [4Fe-4S] clusters, and was highly oxygen-labile. It catalyzed the ATP-dependent reductive dearomatization of BzCoA with 2.3-2.8 ATPs hydrolyzed per two electrons transferred, and preferentially dearomatized methyl-and chloro-substituted analogues in meta- and para-position. NMR analyses revealed that 3-methylbenzoyl-CoA is regioselectively reduced to 3-methyl-1,5-dienoyl-CoA. The unprecedented reductive dechlorination of 4-chloro-BzCoA to BzCoA probably via HCl elimination from a reduced intermediate allowed for the previously unreported growth of T. chlorobenzoica on 4-chloro-benzoate. The heterologous expression platform established in this work enables the production, isolation and characterization of bacterial and archaeal BCR- and BCR-like radical enzymes, for many of which the function has remained unknown.

Brassinosteroids (BRs) play an essential role in plant growth, and BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) family transcription factors integrate a variety of plant signaling pathways. Despite the fact that BRs inhibit nodulation in leguminous plants, how BRs modulate rhizobia-host interactions and nodule morphogenesis is unknown. Here, we show that GmBEHL1, a soybean homolog of Arabidopsis BES1/BZR1 homolog 1 (BEH1), is an interacting partner of Nodule Number Control 1, a transcriptional repressor that mediates soybean nodulation. GmBEHL1 was highly expressed at the basal parts of emerging nodules, and its expression gradually expanded during nodule maturation. The overexpression and downregulation of GmBEHL1 inhibited and enhanced the number of nodules, respectively, in soybean. Intriguingly, alterations in GmBEHL1 expression repressed the expression of genes in the BR biosynthesis pathway, including homologs of Arabidopsis Constitutive Photomorphogenesis and Dwarf and Dwarf 4. We also detected an interaction between GmBEHL1 and GmBIN2, a putative BR-insensitive 2 (BIN2) homolog, in soybean. Moreover, BR treatment reduced the number, but increased the size, of soybean nodules. Our results reveal GmBEHL1 to be a potent gene that integrates BR signaling with nodulation signaling pathways to regulate symbiotic nodulation.

Background and Aims: Pelota (Pelo) are evolutionarily conserved genes reported to be involved in ribosome rescue, cell cycle control and meiotic cell division. However, there is little known about their function in plants. The aim of this study was to elucidate the function of an ethylmethane sulphonate (EMS)-derived mutation of a Pelo-like gene in rice (named Ospelo). Methods: A dysfunctional mutant was used to characterize the function of OsPelo. Analyses of its expression and sub-cellular localization were performed. The whole-genome transcriptomic change in leaves of Ospelo was also investigated by RNA sequencing. Key Results: The Ospelo mutant showed defects in root system development and spotted leaves at early seedling stages. Map-based cloning revealed that the mutation occurred in the putative Pelo gene. OsPelo was found to be expressed in various tissues throughout the plant, and the protein was located in mitochondria. Defence responses were induced in the Ospelo mutant, as shown by enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae, coupled with upregulation of three pathogenesis-related marker genes. In addition, whole-genome transcriptome analysis showed that OsPelo was significantly associated with a number of biological processes, including translation, metabolism and biotic stress response. Detailed analysis showed that activation of a number of innate immunity-related genes might be responsible for the enhanced disease resistance in the Ospelo mutant. Conclusions: These results demonstrate that OsPelo positively regulates root development while its loss of function enhances pathogen resistance by pre-activation of defence responses in rice.

Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilises a type IVb secretion (T4bS) system termed "dot/icm" to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS-like N-terminal domain coupled to a RecA-like C-terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and β, with an αβαβαβ configuration. The existence of α and β conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααβααβ configuration. The two conformers co-exist regardless of the nucleotide-bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug-design to develop more specific antibiotics to tackle Legionnaire's disease. This article is protected by copyright. All rights reserved.

Neutrophils are the first white blood cells to respond to microbes and to limit their invasion of the body. However, the growth of the microbes into colonies often challenges the neutrophils ability to contain them. To study the interactions between neutrophils and microbial colonies, we designed an assay for arranging microbes in clusters of controlled size (i.e. living colloids). The patterned microbes in the living colloid are mechanically trapped inside the wells and fully accessible to neutrophils. Using the assay, we studied the interactions between human neutrophils and Candida albicans and Staphylococcus aureus, two common human pathogens. We also probed the susceptibility of C. albicans colloids to caspofungin, a common antifungal drug.

Serine hydrolases play diverse roles in regulating host-pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here we describe a chemical proteomic screen that identified ten previously uncharacterized S. aureus serine hydrolases that mostly lack human homologs. We termed these enzymes fluorophosphonate-binding hydrolases (FphA-J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of fphB confirmed that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduced infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8-Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O'nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.

Although only a few stem cell-based therapies are currently available to patients, stem cells hold tremendous regenerative potential, and several exciting clinical applications are on the horizon. Biomaterials with tuneable mechanical and biochemical properties can preserve stem cell function in culture, enhance survival of transplanted cells and guide tissue regeneration. Rapid progress with three-dimensional hydrogel culture platforms provides the opportunity to grow patient-specific organoids, and has led to the discovery of drugs that stimulate endogenous tissue-specific stem cells and enabled screens for drugs to treat disease. Therefore, bioengineering technologies are poised to overcome current bottlenecks and revolutionize the field of regenerative medicine.

The host response to viral infection includes the induction of type I interferons and the subsequent upregulation of hundreds of interferon-stimulated genes. Ubiquitin-like protein ISG15 is an interferon-induced protein that has been implicated as a central player in the host antiviral response. Over the past 15 years, efforts to understand how ISG15 protects the host during infection have revealed that its actions are diverse and pathogen-dependent. In this Review, we describe new insights into how ISG15 directly inhibits viral replication and discuss the recent finding that ISG15 modulates the host damage and repair response, immune response and other host signalling pathways. We also explore the viral immune-evasion strategies that counteract the actions of ISG15. These findings are integrated with a discussion of the recent identification of ISG15-deficient individuals and a cellular receptor for ISG15 that provides new insights into how ISG15 shapes the host response to viral infection.

This corrects the article DOI: 10.1038/srep22676.

Light modulates global features of the important human pathogen Acinetobacter baumannii lifestyle including metabolism, tolerance to antibiotics and virulence, most of which depend on the short BLUF-type photoreceptor BlsA. In this work, we show that the ability to circumvent iron deficiency is also modulated by light at moderate temperatures, and disclose the mechanism of signal transduction by showing that BlsA antagonizes the functioning of the ferric uptake regulator (Fur) in a temperature-dependent manner. In fact, we show that BlsA interacts with Fur in the dark at 23 °C, while the interaction is significantly weakened under blue light. Moreover, under iron deprived conditions, expression of Fur-regulated Acinetobactin siderophore genes is only induced in the dark in a BlsA-dependent manner. Finally, growth under iron deficiency is supported in the dark rather than under blue light at moderate temperatures through BlsA. The data is consistent with a model in which BlsA might sequester the repressor from the corresponding operator-promoters, allowing Acinetobactin gene expression. The photoregulation of iron metabolism is lost at higher temperatures such as 30 °C, consistent with fading of the BlsA-Fur interaction at this condition. Overall, we provide new understanding on the functioning of the widespread Fur regulator as well as short-BLUFs.

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.

Furin cleavage of the HIV envelope glycoprotein is an essential step for cell entry that enables formation of well-folded, native-like glycosylated trimers, releases constraints on the fusion peptide, and limits enzymatic processing of the N-glycan shield. Here, we show that a cleavage-independent, stabilized, soluble Env trimer mimic (BG505 NFL.664) exhibits a "closed-form", native-like, prefusion conformation akin to furin-cleaved Env trimers. The crystal structure of BG505 NFL.664 at 3.39 Å resolution with two potent bNAbs also identifies the full epitopes of PGV19 and PGT122 that target the receptor binding site and N332 supersite, respectively. Quantitative site-specific analysis of the glycan shield reveals that native-like glycan processing is maintained despite furin-independent maturation in the secretory pathway. Thus, cleavage-independent NFL Env trimers exhibit quaternary protein and carbohydrate structures similar to the native viral spike that further validate their potential as vaccine immunogen candidates.

In the course of both innate and adaptive immunity, cytidine deaminases within the activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) family modulate immune responses by mutating specific nucleic acid sequences of hosts and pathogens. The evolutionary emergence of these mediators, however, seems to coincide precisely with the emergence of adaptive immunity in vertebrates. Here, we show a family of genes in species within two divergent invertebrate phyla-the echinoderm Strongylocentrotus purpuratus and the brachiopod Lingula anatina-that encode proteins with similarities in amino acid sequence and enzymatic activities to the vertebrate AID/APOBECs. The expression of these invertebrate factors is enriched in tissues undergoing constant, direct interactions with microbes and can be induced upon pathogen challenge. Our findings suggest that AID/APOBEC proteins, and their function in immunity, emerged far earlier than previously thought. Thus, cytidine deamination is probably an ancient innate immune mechanism that predates the protostome/deuterostome divergence.

G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. Many GPCRs are classified as orphan receptors because of the limited knowledge on their ligands and coupling to cellular signaling machineries. Here, we engineer a library of 63 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. Upon stimulation with visible light, we identify activation of canonical cell signaling pathways, including cAMP-, Ca2+-, MAPK/ERK-, and Rho-dependent pathways, downstream of the engineered receptors. For the human pseudogene GPR33, we resurrect a signaling function that supports its hypothesized role as a pathogen entry site. These results demonstrate that substituting unknown chemical activators with a light switch can reveal information about protein function and provide an optically controlled protein library for exploring the physiology and therapeutic potential of understudied GPCRs.

Estimating the size of the viral reservoir is critical for HIV cure strategies. Biomarkers in peripheral circulation may give insights into the establishment of the viral reservoir in compartments not easily accessible. We therefore measured systemic levels of 84 soluble biomarkers belonging to a broad array of immune pathways in acute HIV infection in both antiretroviral therapy-naive (ART-naive) individuals as well as individuals who began ART upon early detection of HIV infection. These biomarkers were measured longitudinally during acute and chronic infection and their relationship to viral reservoir establishment and persistence was assessed. We observed several distinct biomarker pathways induced following HIV infection such as IFN-γ-signaled chemokines, proinflammatory markers, and TNF-α-family members. Levels of several of these factors directly correlated with contemporaneous viral loads and/or frequency of peripheral blood mononuclear cells harboring HIV DNA during acute HIV infection. MCP-1, MIP-3β, sTNFR-II, and IL-10 levels prior to ART associated with HIV DNA levels after 96 weeks of treatment, suggesting a link between early immune signaling events and the establishment and persistence of the viral reservoir during ART. Furthermore, they offer potentially novel tools for gaining insight into relative reservoir size in acutely infected individuals and the potential of associated risks of treatment interruption.

Myeloid leukocytes are essentially involved in both tumor progression and control. We show that neo-adjuvant treatment of mice with an inhibitor of CSF1 receptor (CSF1R), a drug that is used to deplete tumor-associated macrophages, unexpectedly promoted metastasis. CSF1R blockade indirectly diminished the number of NK cells due to a paucity of myeloid cells that provide the survival factor IL-15 to NK cells. Reduction of the number of NK cells resulted in increased seeding of metastatic tumor cells to the lungs but did not impact on progression of established metastases. Supplementation of mice treated with CSF1R-inhibitor with IL-15 restored numbers of NK cells and diminished metastasis. Our data suggest that CSF1R blockade should be combined with administration of IL-15 to reduce the risk of metastasis.

The aging process is associated with diminished colonic health. In this study, we applied an integrative approach to reveal potential interactions between determinants of colonic health in aging C57BL/6J mice. Analysis of gut microbiota composition revealed an enrichment of various potential pathobionts, including Desulfovibrio spp., and a decline of the health-promoting Akkermansia spp. and Lactobacillus spp. during aging. Intraluminal concentrations of various metabolites varied between ages and we found evidence for an increased gut permeability at higher age. Colonic gene expression analysis suggested that during the early phase of aging (between 6 and 12 months), expression of genes involved in epithelial-to-mesenchymal transition and (re)organization of the extracellular matrix were increased. Differential expression of these genes was strongly correlated with Bifidobacterium spp. During the later phase of aging (between 12 and 28 months), gene expression profiles pointed towards a diminished antimicrobial defense and were correlated with an uncultured Gastranaerophilales spp. This study demonstrates that aging is associated with pronounced changes in gut microbiota composition and colonic gene expression. Furthermore, the strong correlations between specific bacterial genera and host gene expression may imply that orchestrated interactions take place in the vicinity of the colonic wall and potentially mediate colonic health during aging.

Streptococcus pneumoniae two-component regulatory systems (TCS) enable adaptation and ensure its maintenance in host environments. This study deciphers the impact of TCS08 on pneumococcal gene expression and its role in metabolic and pathophysiological processes. Transcriptome analysis and real-time PCR demonstrated a regulatory effect of TCS08 on genes involved mainly in environmental information processing, intermediary metabolism, and colonization by S. pneumoniae D39 and TIGR4. Striking examples are genes for fatty acid biosynthesis, genes of the arginine deiminase system, and the psa operon encoding the manganese ABC transport system. In silico analysis confirmed that TCS08 is homologous to Staphylococcus aureus SaeRS, and a SaeR-like binding motif is displayed in the promoter region of pavB, the upstream gene of the tcs08 operon encoding a surface-exposed adhesin. Indeed, PavB is regulated by TCS08 as confirmed by immunoblotting and surface abundance assays. Similarly, pilus-1 of TIGR4 is regulated by TCS08. Finally, in vivo infections using the acute pneumonia and sepsis models showed a strain-dependent effect. Loss of function of HK08 or TCS08 attenuated D39 virulence in lung infections. The RR08 deficiency attenuated TIGR4 in pneumonia, while there was no effect on sepsis. In contrast, lack of HK08 procured a highly virulent TIGR4 phenotype in both pneumonia and sepsis infections. Taken together, these data indicate the importance of TCS08 in pneumococcal fitness to adapt to the milieu of the respiratory tract during colonization.IMPORTANCEStreptococcus pneumoniae interplays with its environment by using 13 two-component regulatory systems and one orphan response regulator. These systems are involved in the sensing of environmental signals, thereby modulating pneumococcal pathophysiology. This study aimed to understand the functional role of genes subject to control by the TCS08. The identified genes play a role in transport of compounds such as sugars or amino acids. In addition, the intermediary metabolism and colonization factors are modulated by TCS08. Thus, TCS08 regulates genes involved in maintaining pneumococcal physiology, transport capacity, and adhesive factors to enable optimal colonization, which represents a prerequisite for invasive pneumococcal disease.

Porcine deltacoronavirus (PDCoV) has recently emerged as an enteric pathogen that can cause serious vomiting and diarrhea in suckling piglets. The first outbreak of PDCoV occurred in the United States in 2014 and was followed by reports of PDCoV in South Korea, China, Thailand, Lao people's Democratic Republic, and Vietnam, leading to economic losses for pig farms and posing considerable threat to the swine industry worldwide. Our previous studies have shown that PDCoV encodes three accessory proteins, NS6, NS7, and NS7a, but the functions of these proteins in viral replication, pathogenesis, and immune regulation remain unclear. Here, we found that ectopic expression of accessory protein NS6 significantly inhibits Sendai virus-induced interferon-β (IFN-β) production, as well as the activation of transcription factors IRF3 and NF-κB. Interestingly, NS6 does not impede the IFN-β promoter activation mediated via key molecules in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, and their downstream molecules MAVS, TBK1, IKKϵ, and IRF3. Further analyses revealed that NS6 is not a RNA-binding protein; however, it interacts with RIG-I/MDA5. This interaction attenuates the binding of double-stranded RNA by RIG-I/MDA5, resulting in the reduction of RLR-mediated IFN-β production. Taken together, our results demonstrate that ectopic expression of NS6 antagonizes IFN-β production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA, revealing a new strategy employed by PDCoV accessory proteins to counteract the host innate antiviral immune response.IMPORTANCECoronavirus accessory proteins are species-specific, and they perform multiple functions in viral pathogenicity and immunity, such as acting as interferon (IFN) antagonists and cell death inducers. Our previous studies have shown that porcine deltacoronavirus (PDCoV) encodes three accessory proteins. Here, we demonstrated for the first time that PDCoV accessory protein NS6 antagonizes IFN-β production by interacting with RIG-I and MDA5 to impede their association with double-stranded RNA. This is an efficient strategy of antagonizing type I IFN production by disrupting the binding of host pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). These findings deepen our understanding of the function of accessory protein NS6 and may direct us toward novel therapeutic targets and lead to the development of more effective vaccines against PDCoV infection.

To enter host cells, herpes simplex virus 1 (HSV-1) initially attaches to cell surface glycosaminoglycans followed by requisite binding to one of several cellular receptors leading to viral internalization. Although virus-receptor interactions have been studied in various cell lines, the contributions of individual receptors to uptake into target tissues such as mucosa, skin or cornea are not well understood. We demonstrated that nectin-1 acts as major receptor for HSV-1 entry into murine epidermis, while HVEM can serve as alternative receptor. Recently, the macrophage receptor with collagenous structure (MARCO) has been described to mediate adsorption of HSV-1 to epithelial cells. Here, we investigated the impact of MARCO on the entry process of HSV-1 into the two major cell types of skin, keratinocytes in the epidermis and fibroblasts in the underlying dermis. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered basal keratinocytes of MARCO-/- epidermis as efficiently as those of control epidermis. In addition, entry into dermal fibroblasts was not impaired in the absence of MARCO. When we treated epidermis, primary keratinocytes or fibroblasts with polyinosinic acid (Poly(I)), a ligand for class A scavenger receptors, HSV-1 entry was strongly reduced. As we observed reducing effects of Poly(I) also in the absence of both MARCO and scavenger receptor A1, we concluded that the inhibitory effects of Poly(I) on HSV-1 infection are not directly linked to class A scavenger receptors. Overall, our results support that HSV-1 entry into skin cells is independent of MARCO.IMPORTANCE During entry into its host cells, the human pathogen herpes simplex virus (HSV) interacts with various cellular receptors. Initially, receptor interaction can mediate cellular adsorption followed by receptor binding that triggers viral internalization. The intriguing question is, which receptors are responsible for the various steps during entry into the natural target tissues of HSV. Previously, we demonstrated the role of nectin-1 as a major and of HVEM as an alternative receptor for HSV-1 to invade murine epidermis. As MARCO has been described to promote infection in skin, we explored the predicted role of MARCO as a receptor that mediates adsorption to epithelial cells. Our infection studies of murine skin cells indicate that the absence of MARCO does not interfere with the efficiency of HSV-1 entry and that the inhibitory effect on viral adsorption by Poly(I), a ligand of MARCO, is independent of MARCO.

Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered that σNS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the Reoviridae family assort and replicate their genomes.

Identifying the causes of high fever syndromes such as Kawasaki disease (KD) remains challenging. To investigate pathogen exposure signatures in suspected pathogen-mediated diseases like KD, we performed immunoglobulin (Ig) profiling using a next-generation sequencing method. After intravenous Ig (IVIG) treatment, we observed disappearance of clonally expanded IgM clonotypes, which were dominantly observed in acute-phase patients. The complementary-determining region 3 (CDR3) sequences of dominant IgM clonotypes in acute-phase patients were commonly observed in other Ig isotypes. In acute-phase KD patients, we identified 32 unique IgM CDR3 clonotypes shared in three or more cases. Furthermore, before the IVIG treatment, the sums of dominant IgM clonotypes in IVIG-resistant KD patients were significantly higher than those of IVIG-sensitive KD patients. Collectively, we demonstrate a novel approach for identifying certain Ig clonotypes for potentially interacting with pathogens involved in KD; this approach could be applied for a wide variety of fever-causing diseases of unknown origin.