Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

Potato gene Y-1 is an N gene homolog that confers cell death upon infection with potato virus Y

ADG2 is a DNA sequence mapped to a resistance (R) gene-rich region at the distal end of chromosome XI in potato (Solanum tuberosum subsp. andigena). The gene, in which ADG2 represents the predicted nucleotide-binding domain (NBS), was cloned and characterized. The coding region of the gene (designated as Y-1) is 6,187 bp long and structurally similar to gene N that confers hypersensitive resistance to Tobacco mosaic virus in Nicotiana spp. Both belong to the TIR-NBS-LRR class of genes and show 57% identity at the amino acid sequence level. The introns of Y-1 were spliced as predicted from the sequence. Y-1 cosegregated with Ry(adg), a gene for extreme resistance to Potato virus Y (PVY) on chromosome XI, as tested in a potato-mapping population and with independent potato cultivars. Leaves of the transgenic potato plants expressing Y-1 under the control of Cauliflower mosaic virus 35S promoter developed necrotic lesions upon infection with PVY, but no significant resistance was observed, and plants were systemically infected with PVY.     

Alternative activation is an innate response to injury that requires CD4+ T cells to be sustained during chronic infection

Alternatively activated macrophages (AAMPhi) are found in abundance during chronic Th2 inflammatory responses to metazoan parasites. Important roles for these macrophages are being defined, particularly in the context of Th2-mediated pathology and fibrosis. However, a full understanding of the requirements for alternative activation, particularly at the innate level, is lacking. We present evidence that alternative activation by the Th2 cytokines IL-4 and IL-13 is an innate and rapid response to tissue injury that takes place even in the absence of an infectious agent. This early response does not require CD4+ Th2 cells because it occurred in RAG-deficient mice. However, class II-restricted CD4+ T cell help is essential to maintain AAMPhi in response to infection, because AAMPhi were absent in RAG-deficient and MHC class II-deficient, but not B cell-deficient mice after chronic exposure to the nematode parasite, Brugia malayi. The absence of AAMPhi was associated with increased neutrophilia and reduced eosinophilia, suggesting that AAMPhi are involved in the clearance of neutrophils as well as the recruitment of eosinophils. Consistent with this hypothesis, AAMPhi show enhanced phagocytosis of apoptotic neutrophils, but not latex beads. Our data demonstrate that alternative activation by type 2 cytokines is an innate response to injury that can occur in the absence of an adaptive response. However, analogous to classical activation by microbial pathogens, Th2 cells are required for maintenance and full activation during the ongoing response to metazoan parasites.     

IL-1 receptor-mediated signal is an essential component of MyD88-dependent innate response to Mycobacterium tuberculosis infection

MyD88, the common adapter involved in TLR, IL-1, and IL-18 receptor signaling, is essential for the control of acute Mycobacterium tuberculosis (MTB) infection. Although TLR2, TLR4, and TLR9 have been implicated in the response to mycobacteria, gene disruption for these TLRs impairs only the long-term control of MTB infection. Here, we addressed the respective role of IL-1 and IL-18 receptor pathways in the MyD88-dependent control of acute MTB infection. Mice deficient for IL-1R1, IL-18R, or Toll-IL-1R domain-containing adaptor protein (TIRAP) were compared with MyD88-deficient mice in an acute model of aerogenic MTB infection. Although primary MyD88-deficient macrophages and dendritic cells were defective in cytokine production in response to mycobacterial stimulation, IL-1R1-deficient macrophages exhibited only a reduced IL-12p40 secretion with unaffected TNF, IL-6, and NO production and up-regulation of costimulatory molecules CD40 and CD86. Aerogenic MTB infection of IL-1R1-deficient mice was lethal within 4 wk with 2-log higher bacterial load in the lung and necrotic pneumonia but efficient pulmonary CD4 and CD8 T cell responses, as seen in MyD88-deficient mice. Mice deficient for IL-18R or TIRAP controlled acute MTB infection. These data demonstrate that absence of IL-1R signal leads to a dramatic defect of early control of MTB infection similar to that seen in the absence of MyD88, whereas IL-18R and TIRAP are dispensable, and that IL-1, together with IL-1-induced innate response, might account for most of MyD88-dependent host response to control acute MTB infection.     

Failure to prove arenavirus infection among the small mammals from an endemic area of Korean hemorrhagic nephrosonephritis.

In the light of recent knowledge on a complex of diseases caused by a new group of viruses, arenaviruses, virological studies largely directed toward small field mammals were undertaken during 1973-1974 aiming at etiological clarification of Korean hemorrhagic nephrosonephritis (KHNN). Specimens were collected in an endemic area of KHNN located north to northeast of Seoul. Virus isolation tests with 299 urine specimens and 131 mite pools recovered from small mammals and 14 acute stage sera from typical cases yielded negative results. Complement-fixation (CF) tests failed to detect antibodies against the antigens of Congo, lymphocytic choriomeningitis (LCM), Tacaribe, and Pichinde viruses among 366 small mammal sera. In addition, CF tests of 59 of the above sera against Apoi and Lassa virus antigens were negative. The results do not support the likelihood of an arenavirus being transmitted among Korean small field mammals, the overwhelming majority of which were Apodemus agrarius. A hypothesis that KHNN is caused by a virus of small field mammal origin was not proved within the technical limit of relatively unsophisticated methods employed herein.     

RNA-seq analysis of the active chick embryo chorioallantoic membrane reveals genes that encode proteins assigned to ion transport and innate immunity

The chicken chorioallantoic membrane (CAM) is an extraembryonic membrane that is vital for the embryo. It undergoes profound cell differentiation between 11 and 15 days of embryonic incubation (EID), which corresponds to the acquisition of its physiological functions. To gain insight into the functional genes that accompany these biological changes, RNA sequencing of the CAM at EID11 and EID15 was performed. Results showed that CAM maturation coincides with the overexpression of 4225 genes, including many genes encoding proteins involved in mineral metabolism, innate immunity, homeostasis, angiogenesis, reproduction, and regulation of hypoxia. Of these genes, some exhibit variability in expression depending on the chicken breed (broiler versus layer breeds). Besides the interest of these results for the poultry sector, the identification of new functional gene candidates opens additional research avenues in the field of developmental biology.     

Pcp4l1 contains an auto-inhibitory element that prevents its IQ motif from binding to calmodulin

Purkinje cell protein 4-like 1 (Pcp4l1) is a small neuronal IQ motif protein closely related to the calmodulin-binding protein Pcp4/PEP-19. PEP-19 interacts with calmodulin via its IQ motif to inhibit calmodulin-dependent enzymes and we hypothesized Pcp4l1 would have similar properties. Surprisingly, full-length Pcp4l1 does not interact with calmodulin in yeast two-hybrid or pulldown experiments yet a synthetic peptide constituting only the IQ motif of Pcp4l1 binds calmodulin and inhibits calmodulin-dependent kinase II. A nine-residue glutamic acid-rich sequence in Pcp4l1 confers these unexpected properties. This element lies outside the IQ motif and its deletion or exchange with the homologous region of PEP-19 restores calmodulin binding. Conversion of a single isoleucine (Ile36) within this motif to phenylalanine, the residue present in PEP-19, imparts calmodulin binding onto Pcp4l1. Moreover, only aromatic amino acid substitutions at position 36 in Pcp4l1 allow binding. Thus, despite their sequence similarities PEP-19 and Pcp4l1 have distinct properties with the latter harboring an element that can functionally suppress an IQ motif. We speculate Pcp4l1 may be a latent calmodulin inhibitor regulated by post-translational modification and/or co-factor interactions.     

A polysaccharide deacetylase from Puccinia striiformis f. sp. tritici is an important pathogenicity gene that suppresses plant immunity

The cell wall of filamentous fungi, comprised of chitin, polysaccharide and glycoproteins, maintains the integrity of hyphae and protect them from defence responses by potential host plants. Here, we report that one polysaccharide deacetylase of Puccinia striiformis f. sp. tritici (Pst), Pst_13661, suppresses Bax‐induced cell death in plants and Pst_13661 is highly induced during early stages of the interaction between wheat and Pst. Importantly, the transgenic wheat expressing the RNA interference (RNAi) construct of Pst_13661 exhibits high resistance to major Pst epidemic races CYR31, CYR32 and CYR33 by inhibiting growth and development of Pst, indicating that Pst_13661 is an available pathogenicity factor and is a potential target for generating broad‐spectrum resistance breeding material of wheat. It forms a homo‐polymer and has high affinity for chitin and germ tubes of Pst compared with the control. Besides, Pst_13661 suppresses chitin‐induced plant defence in plants. Hence, we infer that Pst_13661 may modify the fungal cell wall to prevent recognition by apoplastic surveillance systems in plants. This study opens new approaches for developing durable disease‐resistant germplasm by disturbing the growth and development of fungi and develops novel strategies to control crop diseases.     

Epstein-Barr virus BZLF1 gene, a switch from latency to lytic infection, is expressed as an immediate-early gene after primary infection of B lymphocytes

We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.     

The Drosophila inhibitor of apoptosis protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.     

The gene of retroviral origin Syncytin 1 is specific to hominoids and is inactive in Old World monkeys

Syncytin 1 is one of the best known examples of recent acquisition of a new gene from an endogenous retrovirus (HERV) in the human genome and has been implicated in placental physiology. Within primates, Syncytin 1 is conserved in all hominoids but has not been characterized in Old World monkeys (OWMs). In this study, we investigated the status of Syncytin 1 in 14 hominoid and OWM species. We show that although the HERV-W provirus responsible for the origin of this gene was present in the genome of the most recent common ancestor of hominoids and OWMs, Syncytin 1 is inactive in OWMs. In addition, we were able to determine that the evolution of Syncytin 1 in hominoids involved an accumulation of amino acid changes and showed signatures of both positive and purifying selection. Our results indicate that Syncytin 1 is indeed a hominoid-specific gene and illustrate the complex and dynamic process associated with the origin of new genes.     

Respiratory syncytial virus infection is associated with an altered innate immunity and a heightened pro-inflammatory response in the lungs of preterm lambs

Introduction

Factors explaining the greater susceptibility of preterm infants to severe lower respiratory infections with respiratory syncytial virus (RSV) remain poorly understood. Fetal/newborn lambs are increasingly appreciated as a model to study key elements of RSV infection in newborn infants due to similarities in lung alveolar development, immune response, and susceptibility to RSV. Previously, our laboratory demonstrated that preterm lambs had elevated viral antigen and developed more severe lesions compared to full-term lambs at seven days post-infection. Here, we compared the pathogenesis and immunological response to RSV infection in lungs of preterm and full-term lambs.

Methods

Lambs were delivered preterm by Caesarian section or full-term by natural birth, then inoculated with bovine RSV (bRSV) via the intratracheal route. Seven days post-infection, lungs were collected for evaluation of cytokine production, histopathology and cellular infiltration.

Results

Compared to full-term lambs, lungs of preterm lambs had a heightened pro-inflammatory response after infection, with significantly increased MCP-1, MIP-1α, IFN-γ, TNF-α and PD-L1 mRNA. RSV infection in the preterm lung was characterized by increased epithelial thickening and periodic acid-Schiff staining, indicative of glycogen retention. Nitric oxide levels were decreased in lungs of infected preterm lambs compared to full-term lambs, indicating alternative macrophage activation. Although infection induced significant neutrophil recruitment into the lungs of preterm lambs, neutrophils produced less myeloperoxidase than those of full-term lambs, suggesting decreased functional activation.

Conclusions

Taken together, our data suggest that increased RSV load and inadequate immune response may contribute to the enhanced disease severity observed in the lungs of preterm lambs.     

The IL-27 receptor (WSX-1) is an inhibitor of innate and adaptive elements of type 2 immunity

Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1-/- mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.     

Human host genetic factors in mycobacterial and Salmonella infection: lessons from single gene disorders in IL-12/IL-23-dependent signaling that affect innate and adaptive immunity

Interleukin (IL)-12/IL-23 signal transduction-deficient individuals with genetic defects in IL12RB1 or IL12B often suffer from unusual mycobacterial and Salmonella infections. Here we discuss recent questions that have arisen from clinical observations that cast doubt on the necessity of IL-12/IL-23 signaling in controlling infections with intracellular bacteria. Alternative IL-12/IL-23-dependent, interferon-gamma-independent pathways of immunity to intracellular bacteria are also discussed.     

Isolation of an active gene encoding human hnRNP protein A1. Evidence for alternative splicing

Heterogeneous nuclear ribonucleoprotein (hnRNP) core protein A1 is a major component of mammalian hnRNP 40 S particles. We describe the structure of an active A1 gene and report on the partial characterization of the A1 gene family. About 30 A1-specific sequences are present per haploid human genome: 15 such sequences were isolated from a human genomic DNA library. Many corresponded to pseudogenes of the processed type but by applying a selection for actively transcribed regions we isolated an active A1 gene. The gene spans a region of 4.6 x 10(3) base-pairs and it is split into ten exons that encode the 320 amino acid residues of the protein. The amino acid sequence derived from the exon sequences is identical with that deduced from cDNA and reported for the protein. One intron exactly separates the two structural domains that constitute the protein. Each of the two RNA-binding domains in protein A1 is encoded by one exon. Experimental evidence indicates that the A1 gene can encode for more than one protein by alternative splicing. The gene is preceded by a strong promoter that contains at least two CCAAT boxes and two possible Sp1 binding sites, but it lacks a TATA box.     

Exit of Plasmodium sporozoites from oocysts is an active process that involves the circumsporozoite protein

Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.     

DNA sequence of an amber replication mutant indicates that a 29 kd protein is the product of the F plasmid replication gene

Summary DNA sequencing shows that the mutational alteration resulting from an amber-suppressible replication-defective mutation of F plasmid is a single base pair change from C:G to T:A which yields an amber codon in the coding frame for a 29 kd polypeptide located in the minimal replication region. We thus identified the gene indispensable for F DNA replication as the coding frame which encodes a 29 kb polypeptide. We will designate this gene repA.Abbreviations kb kilobase pairs - bp base pair - kd kilodaltons     

Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor

The micrographic phytopathogen Botrytis cinerea causes gray mold diseases in a large number of dicotyledonous crop plants and ornamentals. Colonization of host tissue is accompanied by rapid killing of plant cells ahead of the growing hyphen, probably caused by secretion of nonspecific phytotoxins, e.g., the sesquiterpene botrydial. Although all pathogenic strains tested so far had been shown to secrete botrydial and although the toxin causes comparable necrotic lesions as infection by the fungus, the role of botrydial in the infection process has not been elucidated so far. Here, we describe the functional characterization of bcbot1, encoding a P450 monooxygenase and provide evidence that it is involved in the botrydial pathway, i.e., it represents the first botrydial biosynthetic gene identified. We show that bcbot1 is expressed in planta and that expression in vitro and in planta is controlled by an alpha-subunit of a heterotrimeric GTP-binding protein, BCG1. Deletion of bcbot1 in three standard strains of B. cinerea shows that the effect on virulence (on several host plants) is strain-dependent; only deletion in one of the strains (T4) led to reduced virulence.