Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3X (DDX3X) is an ATP-dependent RNA helicase, In addition to involvement of eukaryotic gene expression regulation, mammalian DDX3X has recently been found to regulate IFN-β production via the adaptor MAVS mediated cascade signaling. In our studies, we demonstrated that chicken DDX3X (chDDX3X) is also involved in the IFN-β regulation, and demonstrated that chDDX3X regulated IFN-β via an essential adaptor chicken stimulator of IFN genes (chSTING). We found that chDDX3X overexpression in DF-1 cells induced expression of IFN-β and inhibited avian influenza virus (AIV) or Newcastle disease virus (NDV) replication. Knockdown of chDDX3X decreased the production of IFN-β induced by RNA analog polyinosinic-polycytidylic acid and increased viral yield. Furthermore, chicken chDDX3X was identified as a potential chSTING-interacting protein by endogenous co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). And exogenous Co-IP in transfected cells with or without virus-stimulations cells further confirmed the interaction between chDDX3X and chSTING. With the gene overexpression and RNA interference studies, the chDDX3X was confirmed to be located upstream of chSTING and activate IFN-β via the chSTING-chTBK1-chIRF7-IFN-β signaling axis. In brief, our results suggest that chDDX3X is an important IFN-β mediator and is involved in RNA- and RNA virus-mediated chDDX3X-chSTING-IFN-β signaling pathway.
Bats are important hosts for various zoonotic viral diseases. However, they rarely show signs of disease infection with such viruses. As the first line for virus control, the innate immune system of bats attracted our full attention. In this study, the Tadarida brasiliensis MDA5 gene (batMDA5), a major sensor for anti-RNA viral infection, was first cloned, and its biological functions in antiviral innate immunity were identified. Bioinformatics analysis shows that the amino acid sequence of batMDA5 is poorly conserved among species, and it is evolutionarily closer to humans. The mRNA of batMDA5 was significantly upregulated in Newcastle disease virus (NDV), avian influenza virus (AIV), and vesicular stomatitis virus (VSV)-infected bat TB 1 Lu cells. Overexpression of batMDA5 could activate IFNβ and inhibit vesicular stomatitis virus (VSV-GFP) replication in TB 1 Lu cells, while knockdown of batMDA5 yielded the opposite result. In addition, we found that the CARD domain was essential for MDA5 to activate IFNβ by constructing MDA5 domain mutant plasmids. These results indicated that bat employs a conserved MDA5 gene to trigger anti-RNA virus innate immune response. This study helps understand the biological role of MDA5 in innate immunity during evolution.
A bacterial strain named STR B was isolated from soil,which could be cultivated on some media,such as SFM,LB,EMB,PDA,MM and MacC agar.It was gram negative and could be viewed without flagellas under electron microscope.The strain could not ferment most of sugars in the biochemical test and it was identified as Acinetobacter calcoaceticus with the gram negative identification card(GNI)of bioMerieux Vitek Company.Two pairs of primers were designed for PCR of its 16S rRNA.The sequence analysis results of 16S rRNA sequence with online software showed that the STR B belonged to Acinetobacter johnsonii,not a strain of Acinetobacter calcoaceticus.
Chickens are the natural host of Newcastle disease virus (NDV) and avian influenza virus (AIV). The discovery that the RIG-I gene, the primary RNA virus pattern recognition receptor (PRR) in mammals, is naturally absent in chickens has directed attention to studies of chicken RNA PRRs and their functions in antiviral immune responses. Here, we identified Asp-Glu-Ala-Asp (DEAD)-box helicase 1 (DDX1) as an essential RNA virus PRR in chickens and investigated its functions in anti-RNA viral infections. The chDDX1 gene was cloned, and cross-species sequence alignment and phylogenetic tree analyses revealed high conservation of DDX1 among vertebrates. A quantitative RT-PCR showed that chDDX1 mRNA are widely expressed in different tissues in healthy chickens. In addition, chDDX1 was significantly upregulated after infection with AIV, NDV, or GFP-expressing vesicular stomatitis virus (VSV-GFP). Overexpression of chDDX1 in DF-1 cells induced the expression of IFN-β, IFN-stimulated genes (ISGs), and proinflammatory cytokines; it also inhibited NDV and VSV replications. The knockdown of chDDX1 increased the viral yield of NDV and VSV and decreased the production of IFN-β, which was induced by RNA analog polyinosinic-polycytidylic acid (poly[I:C]), by AIV, and by NDV. We used a chicken IRF7 (chIRF7) knockout DF-1 cell line in a series of experiments to demonstrate that chDDX1 activates IFN signaling via the chIRF7 pathway. Finally, an in-vitro pulldown assay showed a strong and direct interaction between poly(I:C) and the chDDX1 protein, indicating that chDDX1 may act as an RNA PRR during IFN activation. In brief, our results suggest that chDDX1 is an important mediator of IFN-β and is involved in RNA- and RNA virus-mediated chDDX1-IRF7-IFN-β signaling pathways.
The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane alpha-helical spanners, belonging to the drug:H(+) antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [(3)H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.
Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli (E. coli) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δhns mutant (MG1655Δhns) of the E. coli K-12 strain MG1655 was obtained. The Δhns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δhns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.
Reassortant variant viruses generated between 2009 H1N1 pandemic influenza virus [A(H1N1)pdm09] and endemic swine influenza viruses posed a potential risk to humans. Surprisingly, genetic analysis showed that almost all of these variant viruses contained the M segment from A(H1N1)pdm09, which originated from Eurasian avian-like swine influenza viruses. Studies have shown that the A(H1N1)pdm09 M gene is critical for the transmissibility and pathogenicity of the variant viruses. However, the M gene encodes two proteins, M1 and M2, and which of those plays a more important role in virus pathogenicity remains unknown. In this study, the M1 and M2 genes of A(H1N1)pdm09 were replaced with those of endemic H3N2 swine influenza virus, respectively. The chimeric viruses were rescued and evaluated in vitro and in mice. Both M1 and M2 of H3N2 affected the virus replication in vitro. In mice, the introduction of H3N2 M1 attenuated the chimeric virus, where all the mice survived from the infection, compared with the wild type virus that caused 100 % mortality. However, the chimeric virus containing H3N2 M2 was still virulent to mice, and caused 16.6% mortality, as well as similar body weight loss to the wild type virus infected group. Compared with the wild type virus, the chimeric virus containing H3N2 M1 induced lower levels of inflammatory cytokines and higher levels of anti-inflammatory cytokines, whereas the chimeric virus containing H3N2 M2 induced substantial pro-inflammatory responses, but higher levels of anti-inflammatory cytokines. The study demonstrated that Eurasian avian-like M1 played a more important role than M2 in the pathogenicity of A(H1N1)pdm09 in mice.
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