RIG-I and MDA5 are two key pattern recognition receptors that sense RNA virus invasion, but RIG-I is absent in chickens. Although chickens have intact MDA5, the genes downstream of chicken MDA5 (chMDA5) that may mediate antiviral response are not well studied. We compared the transcriptional profile of chicken embryonic fibroblasts (DF1) transfected with chMDA5, and poly(I:C), using RNA-seq. Transfected chMDA5 and poly(I:C) in DF1 cells were associated with the marked induction of many antiviral innate immune genes compared with control. Interestingly, nine interferon-stimulated genes (ISGs) were listed in the top 15 upregulated genes by chMDA5 and poly(I:C) transfection. We used real-time PCR to confirm the upregulation of the nine ISGs, namely, MX1, IFI6, IFIT5, RSAD2, OASL, CMPK2, HELZ2, EPSTI1, and OLFML1, by chMDA5 and poly(I:C) transfection in DF1 cells. However, avian influenza virus H5N6 infection only increased MX1, IFI6, IFIT5, RSAD2, and OASL expression levels. Further study showed that the overexpression of these five genes could significantly inhibit H5N6 virus replication. These results provide some insights into the gene expression pattern induced by chMDA5, which would be beneficial for understanding and identifying innate immune genes of chicken that may lead to new antiviral therapies.
M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation.
Influenza A virus (IVA) has been continuously causing pandemics in several animal hosts and has become a worldwide public health threat. Currently, antiviral drugs have become associated with a lot of side effects and limited activity against emerging drug-resistant influenza viruses. Therefore, the development of novel antiviral drugs is of great importance. In this study, we synthesized a kind of carbon dots (CDs) with high dispersibility from glycyrrhizic acid (GA) using a simple dry heating method. Compared with glycyrrhizic acid alone, GA-CDs exhibit superior solubility and significantly improve the antiviral property against IVA. Investigation of the mechanism revealed that GA-CDs act against IVA mainly by inhibiting viral internalization, replication of the viral genome, neuraminidase activity, and host inflammatory responses. More importantly, in a mouse model, GA-CDs can significantly alleviate the clinical symptoms and decrease mortality and lung viral titers. In vitro and in vivo experiments demonstrate that GA-CDs possess extraordinary therapeutic effects; therefore, we propose that GA-CDs may be a promising alternative therapy for IVA infection.
Influenza A virus (IAV) poses a serious threat to human life and property. The IAV matrix protein 2 (M2) is significant in viral budding. Increasing studies have proven the important roles of host factors in IAV replication. In this study, immunoprecipitation combined with mass spectrometry revealed that the host protein tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG), which belongs to the 14-3-3 protein scaffold family, interacts with M2. Their interactions were further confirmed by co-immunoprecipitation (Co-IP), immunofluorescence, and confocal microscopy of virus-infected HeLa cells. Moreover, we constructed YWHAG-KO and YWHAG-overexpressing cells and found that YWHAG knockout significantly increased viral production, whereas its overexpression reduced the titer of virus progeny. Therefore, YWHAG is a negative regulatory factor during IAV infection. Further, YWHAG knockout or overexpression had no effect on the binding, entry, or viral RNA replication in the early stages of the virus life cycle. On the contrary, it impaired the release of virions at the plasma membrane as determined using transmission electron microscopy and suppressed the M2-mediated budding of the influenza virus. Importantly, the H158F mutation of YWHAG was found to affect interaction with M2 and its budding. Collectively, our work demonstrates that YWHAG is a novel cellular regulator that targets and mediates the interaction and release of M2.
Avian influenza A (H7N9) viruses emerged in China in 2013 and caused a zoonotic disease associated with a high case-fatality ratio of more than 30%. Transcriptional profiles obtained using animal models reveal host responses to the disease, thereby providing insights into disease pathogenesis. Therefore, we aimed to characterize the host responses of the H7N9 virus infected-mouse lungs in this study. First, we isolated an avian-originated H7N9 strain, which was shown to be highly pathogenic to both chickens and mice. Genomic analysis results suggested that a 12-nucleotide-insertion was present at the hemagglutinin cleavage site, and both the hemagglutinin and neuraminidase genes belonged to the Yangtze River Delta lineage. RNA sequencing results revealed 566 differentially expressed genes in the H7N9-infected lungs. Moreover, transcriptome analysis revealed that over-activated antiviral signals and intense interferon-stimulated gene products possibly contributed to the high virulence of the virus in mice. Importantly, lung concentrations of inflammatory cytokines, including interleukin-1β and interleukin-6, interferon-β, and tumor necrosis factor-α, were upregulated in response to H7N9 virus infection. Overall, the present study provided a comprehensive understanding of H7N9 virus pathogenicity and correlated host immune responses.
RIG-I and MDA5 are two key pattern recognition receptors that sense the invasion of RNA viruses and initiate type I interferon (IFN) response. Although these receptors are generally conserved in vertebrates, RIG-I is absent in chickens, whereas MDA5 is present. Chicken MDA5 (chMDA5) plays a pivotal role in sensing the invasion of RNA viruses into cells. However, unlike mammalian MDA5, where there are in-depth and extensive studies, regulation of the chMDA5-mediated signaling pathway remains unexplored. In this study, we performed a pulldown assay and mass spectrometry analysis to identify chicken proteins that could interact with the N terminal of chMDA5 (chMDA5-N) that contained two CARDs responsible for binding of the well-known downstream adaptor MAVS. We found that 337 host proteins could potentially interact with chMDA5-N, which were integrated to build a chMDA5-N–host association network and analyzed by KEGG pathway and Gene Ontology annotation. Results of our analysis revealed that diverse cellular processes, such as RNA binding and transport and protein translation, ribosome, chaperones, and proteasomes are critical cellular factors regulating the chMDA5-mediated signaling pathway. We cloned 64 chicken genes to investigate their effects on chMDA5-mediated chicken IFN-β production and confirmed the association of chicken DDX5, HSPA8, HSP79, IFIT5, PRDX1, and hnRNPH2 with chMDA5-N. In particular, we found that chicken hnRNPH2 impairs the association between chMDA5-N and MAVS and thus acts as a check on the chMDA5-mediated signaling pathway. To our knowledge, this study is the first to analyze the chicken MDA5–host interactome, which provides fundamental but significant insights to further explore the mechanism of chicken MDA5 signaling regulation in detail.
H5N6, the highly pathogenic avian influenza A virus (IAV) of clade 2.3.4.4, causes global outbreaks in poultry. H5N6 has become the dominant IAV subtype in waterfowls and causes human infections with high mortality rates. Here, we isolated two strains of H5N6, XGD and JX, from chickens and ducks, respectively. Growth kinetics were evaluated in duck embryo fibroblasts, chicken embryo fibroblasts, Madin-Darby canine kidney cells, and A549 lung carcinoma cells. Receptor binding specificity was analyzed via sialic acid–binding activity assay. The virulence of each strain was tested in BALB/c mice, and recombinant viruses were constructed via reverse genetics to further analyze the pathogenicity. The two strains showed no significant differences in growth kinetics in vitro; however, JX was more virulent in mice than XGD. We also identified 13 mutations in six viral proteins of the two strains through genetic analysis. Our study showed that the NS1 protein played a crucial role in enhancing the virulence of JX. Specifically, the amino acid 139D in NS1 contributed to the high pathogenicity. Therefore, 139D in NS1 might provide insight into the underlying mechanism of IAV adaptation in mammals.
The RNA-splicing ligase RNA 2′,3′-cyclic phosphate and 5′-OH ligase (RTCB) is a catalytic subunit of the tRNA-splicing ligase complex, which plays an essential role in catalyzing tRNA splicing and modulating the unfolded protein response. However, the function of RTCB in influenza A virus (IAV) replication has not yet been described. In this study, RTCB was revealed to be an IAV-suppressed host factor that was significantly downregulated during influenza virus infection in several transformed cell lines, as well as in primary human type II alveolar epithelial cells, and its knockout impaired the propagation of the IAV. Mechanistically, RTCB depletion led to a robust elevation in the levels of type I and type III IFNs and proinflammatory cytokines in response to IAV infection, which was confirmed by RTCB overexpression studies. Lastly, RTCB was found to compete with DDX21 for RNA helicase DDX1 binding, attenuating the DDX21-DDX1 association and thus suppressing the expression of IFN and downstream IFN-stimulated genes. Our study indicates that RTCB plays a critical role in facilitating IAV replication and reveals that the RTCB-DDX1 binding interaction is an important innate immunomodulator for the host to counteract viral infection.
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