ABSTRACT
Many annotated long noncoding RNAs (lncRNAs) contain small open reading frames (sORFs), some of which have been demonstrated to encode small proteins or micropeptides with fundamental biological importance. However, functions of lncRNAs-encoded small proteins or micropeptides in viral pathogenesis remain largely unexplored. Here, we identified a 110-amino acid small protein as a key regulator of influenza A virus (IAV) replication. This small protein that we call PESP was encoded by the putative lncRNA PCBP1-AS1. It was observed that both PCBP1-AS1 and PESP were significantly upregulated by IAV infection. Furthermore, they were markedly induced by treatment with either type I or type III interferon. Overexpression of either PCBP1-AS1 or PESP alone significantly enhanced IAV replication. In contrast, shRNA-mediated knockdown of PCBP1-AS1 or CRISPR/Cas9-mediated knockout of PESP markedly inhibited the viral production. Moreover, the targeted deletion or mutation of the sORF within the PCBP1-AS1 transcript, which resulted in the disruption of PESP expression, significantly diminished the capacity of PCBP1-AS1 to enhance IAV replication, underscoring the indispensable role of PESP in the facilitation of IAV replication by PCBP1-AS1. Interestingly, overexpression of PESP enhanced the IAV-induced autophagy by increasing the expression of ATG7, an essential autophagy effector enzyme. We also found that the 7-22 amino acids at the N-terminus of PESP were crucial for its functionality in modulating ATG7 expression and action as an enhancer of IAV replication. Additionally, HSP90AA1, a protein identified previously as a facilitator of autophagy, was found to interact with PESP, resulting in the stabilization of PESP and consequently an increase in the production of IAV. These data reveal a critical lncRNA-encoded small protein that is induced and exploited by IAV during its infection, and provide a significant insight into IAV-host interaction network.
Subject(s)
Autophagy , Influenza A virus , RNA, Long Noncoding , RNA-Binding Proteins , Virus Replication , Virus Replication/physiology , Humans , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Influenza A virus/genetics , Influenza A virus/metabolism , Influenza, Human/virology , Influenza, Human/metabolism , Influenza, Human/genetics , A549 Cells , Animals , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , DNA-Binding ProteinsABSTRACT
African swine fever (ASF) is an infectious disease with high mortality caused by African swine fever virus (ASFV), which poses a great threat to the global swine industry. ASFV has evolved multiple strategies to evade host antiviral innate immunity by perturbing inflammatory responses and interferon production. However, the molecular mechanisms underlying manipulation of inflammatory responses by ASFV proteins are not fully understood. Here, we report that A137R protein of ASFV is a key suppressor of host inflammatory responses. Ectopic expression of ASFV A137R in HEK293T cells significantly inhibited the activation of IL-8 and NF-κB promoters triggered by Sendai virus (SeV), influenza A virus (IAV), or vesicular stomatitis virus (VSV). Accordingly, forced A137R expression caused a significant decrease in the production of several inflammatory cytokines such as IL-8, IL-6 and TNF-α in the cells infected with SeV or IAV. Similar results were obtained from experiments using A137R overexpressing PK15 and 3D4/21 cells infected with SeV or VSV. Furthermore, we observed that A137R impaired the activation of MAPK and NF-κB signaling pathways, as enhanced expression of A137R significantly decreased the phosphorylation of JNK, p38 and p65 respectively upon viral infection (SeV or IAV) and IL-1ß treatment. Mechanistically, we found that A137R interacted with MyD88, and dampened MyD88-mediated activation of MAPK and NF-κB signaling. Together, these findings uncover a critical role of A137R in restraining host inflammatory responses, and improve our understanding of complicated mechanisms whereby ASFV evades innate immunity.
Subject(s)
African Swine Fever Virus , African Swine Fever , Swine Diseases , Animals , Swine , Humans , NF-kappa B/metabolism , African Swine Fever Virus/genetics , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Interleukin-8/metabolism , HEK293 CellsABSTRACT
Cancer is the leading cause of death in both humans and companion animals. Canine mammary tumor is an important disease with a high incidence and metastasis rate, and its poor prognosis remains a serious clinical challenge. C6 ceramide is a short-chain sphingolipid metabolite with powerful potential as a tumor suppressor. However, the specific impact of C6 ceramide on canine mammary cancer remains unclear. However, the effects of C6 ceramide in canine mammary cancer are still unclear. Therefore, we investigated the role of C6 ceramide in the progress of canine mammary cancer and explored its potential mechanism. C6 ceramide inhibited cell growth by regulating the cell cycle without involving apoptosis. Additionally, C6 ceramide inhibited the migration and invasion of CHMp cells. In vivo, C6 ceramide decreased tumor growth and metastasis in the lungs without side effects. Further investigation found that the knockdown of EGR3 expression led to a noticeable increase in proliferation and migration by upregulating the expressions of pJAK1 and pSTAT3, thus activating the JAK1/STAT3 signaling pathway. In conclusion, C6 ceramide inhibits canine mammary cancer growth and metastasis by targeting EGR3 through the regulation of the JAK1/STAT3 signaling pathway. This study implicates the mechanisms underlying the anti-tumor activity of C6 ceramide and demonstrates the potential of EGR3 as a novel target for treating canine mammary cancer.
ABSTRACT
Amidst increasing concern about antibiotic resistance resulting from the overuse of antibiotics, there is a growing interest in exploring alternative agents. One such agent is citric acid, an organic compound commonly used for various applications. Our research findings indicate that the inclusion of citric acid can have several beneficial effects on the tight junctions found in the mouse intestine. Firstly, the study suggests that citric acid may contribute to weight gain by stimulating the growth of intestinal epithelial cells (IE-6). Citric acid enhances the small intestinal villus-crypt ratio in mice, thereby promoting intestinal structural morphology. Additionally, citric acid has been found to increase the population of beneficial intestinal microorganisms, including Bifidobacterium and Lactobacillus. It also promotes the expression of important protein genes such as occludin, ZO-1, and claudin-1, which play crucial roles in maintaining the integrity of the tight junction barrier in the intestines. Furthermore, in infected IEC-6 cells with H9N2 avian influenza virus, citric acid augmented the expression of genes closely associated with the influenza virus infection. Moreover, it reduces the inflammatory response caused by the viral infection and thwarted influenza virus replication. These findings suggest that citric acid fortifies the intestinal tight junction barrier, inhibits the replication of influenza viruses targeting the intestinal tract, and boosts intestinal immune function.
Subject(s)
Influenza A Virus, H9N2 Subtype , Influenza, Human , Animals , Mice , Humans , Citric Acid/pharmacology , Citric Acid/metabolism , Influenza, Human/metabolism , Intestines/microbiology , Intestinal Mucosa/metabolism , Tight Junctions/metabolism , ImmunityABSTRACT
Autophagy-related protein 7 (ATG7) is an essential autophagy effector enzyme. Although it is well known that autophagy plays crucial roles in the infections with various viruses including influenza A virus (IAV), function and underlying mechanism of ATG7 in infection and pathogenesis of IAV remain poorly understood. Here, in vitro studies showed that ATG7 had profound effects on replication of IAV. Depletion of ATG7 markedly attenuated the replication of IAV, whereas overexpression of ATG7 facilitated the viral replication. ATG7 conditional knockout mice were further employed and exhibited significantly resistant to viral infections, as evidenced by a lower degree of tissue injury, slower body weight loss, and better survival, than the wild type animals challenged with either IAV (RNA virus) or pseudorabies virus (DNA virus). Interestingly, we found that ATG7 promoted the replication of IAV in autophagy-dependent and -independent manners, as inhibition of autophagy failed to completely block the upregulation of IAV replication by ATG7. To determine the autophagy-independent mechanism, transcriptome analysis was utilized and demonstrated that ATG7 restrained the production of interferons (IFNs). Loss of ATG7 obviously enhanced the expression of type I and III IFNs in ATG7-depleted cells and mice, whereas overexpression of ATG7 impaired the interferon response to IAV infection. Consistently, our experiments demonstrated that ATG7 significantly suppressed IRF3 activation during the IAV infection. Furthermore, we identified long noncoding RNA (lncRNA) GAPLINC as a critical regulator involved in the promotion of IAV replication by ATG7. Importantly, both inactivation of IRF3 and inhibition of IFN response caused by ATG7 were mediated through control over GAPLINC expression, suggesting that GAPLINC contributes to the suppression of antiviral immunity by ATG7. Together, these results uncover an autophagy-independent mechanism by which ATG7 suppresses host innate immunity and establish a critical role for ATG7/GAPLINC/IRF3 axis in regulating IAV infection and pathogenesis.
Subject(s)
Influenza A virus , Influenza, Human , Virus Diseases , Animals , Humans , Mice , Immunity, Innate , Interferons , Virus ReplicationABSTRACT
Cupriavidus sp. L7L synthesizes a high content of ductile polyhydroxyalkanoate. However, during fermentation, the medium's viscosity gradually increases, eventually reaching a level similar to 93 % glycerol, leading to fermentation termination and difficulties in cell harvest. A non-mucoid variant was isolated from a mini-Tn5 mutant library with the transposon inserted at the promoter sequence upstream of the wcaJ gene. Deletion of wcaJ eliminated the mucoid-colony appearance. The complementation experiment confirmed the association between wcaJ gene expression and mucoid-colony formation. Additionally, the wild-type strain exhibited a faster specific growth rate than the deletion strain using levulinate (Lev) as a carbon source. In fed-batch fermentation, Cupriavidus sp. L7L∆wcaJ showed similar PHA content and monomer composition to the wild-type strain. However, the extended fermentation time resulted in a 42 % increase in PHA concentration. After fed-batch fermentation, the deletion strain's medium had only 8.75 % of the wild-type strain's extracellular polymeric substance content. Moreover, the deletion strain's medium had a much lower viscosity (1.04 mPa·s) than the wild-type strain (194.7 mPa·s), making bacterial cell collection easier through centrifugation. In summary, Cupriavidus sp. L7L∆wcaJ effectively addressed difficulties in cell harvest, increased PHA production, and Lev-to-PHA conversion efficiency, making these characteristics advantageous for industrial-scale PHA production.
Subject(s)
Cupriavidus necator , Cupriavidus , Polyhydroxyalkanoates , Cupriavidus/genetics , Cupriavidus/metabolism , Extracellular Polymeric Substance Matrix/metabolism , Gene Deletion , Fermentation , Cupriavidus necator/metabolismABSTRACT
Effective viral clearance requires fine-tuned immune responses to minimize undesirable inflammatory responses. Circular RNAs (circRNAs) are a class of non-coding RNAs that are abundant and highly stable, formed by backsplicing pre-mRNAs, and expressed ubiquitously in eukaryotic cells, emerging as critical regulators of a plethora of signaling pathways. Recent progress in high-throughput sequencing has enabled a better understanding of the physiological and pathophysiological functions of circRNAs, overcoming the obstacle of the sequence overlap between circRNAs and their linear cognate mRNAs. Some viruses also encode circRNAs implicated in viral replication or disease progression. There is increasing evidence that viral infections dysregulate circRNA expression and that the altered expression of circRNAs is critical in regulating viral infection and replication. circRNAs were shown to regulate gene expression via microRNA and protein sponging or via encoding small polypeptides. Recent studies have also highlighted the potential role of circRNAs as promising diagnostic and prognostic biomarkers, RNA vaccines and antiviral therapy candidates due to their higher stability and lower immunogenicity. This review presents an up-to-date summary of the mechanistic involvement of circRNAs in innate immunity against viral infections, the current understanding of their regulatory roles, and the suggested applications.
Subject(s)
RNA, Circular , Virus Diseases , Humans , RNA, Circular/genetics , Immunity, Innate , Virus Diseases/genetics , Disease Progression , Eukaryotic CellsABSTRACT
This study identifies interleukin-6 (IL-6)-independent phosphorylation of STAT3 Y705 at the early stage of infection with several viruses, including influenza A virus (IAV). Such activation of STAT3 is dependent on the retinoic acid-induced gene I/mitochondrial antiviral-signaling protein/spleen tyrosine kinase (RIG-I/MAVS/Syk) axis and critical for antiviral immunity. We generate STAT3Y705F/+ knockin mice that display a remarkably suppressed antiviral response to IAV infection, as evidenced by impaired expression of several antiviral genes, severe lung tissue injury, and poor survival compared with wild-type animals. Mechanistically, STAT3 Y705 phosphorylation restrains IAV pathogenesis by repressing excessive production of interferons (IFNs). Blocking phosphorylation significantly augments the expression of type I and III IFNs, potentiating the virulence of IAV in mice. Importantly, knockout of IFNAR1 or IFNLR1 in STAT3Y705F/+ mice protects the animals from lung injury and reduces viral load. The results indicate that activation of STAT3 by Y705 phosphorylation is vital for establishment of effective antiviral immunity by suppressing excessive IFN signaling induced by viral infection.
Subject(s)
Influenza A virus , Orthomyxoviridae Infections , STAT3 Transcription Factor , Animals , Mice , Antiviral Agents , Immunity, Innate , Interferons , Receptors, Interferon , Signal Transduction , Orthomyxoviridae Infections/immunology , STAT3 Transcription Factor/immunologyABSTRACT
The clinical benefits of targeting programmed death-ligand 1 (PD-L1) in various cancers represent a strategy for the treatment of immunosuppressive diseases. Here, it was demonstrated that the expression levels of PD-L1 in cells were greatly upregulated in response to H1N1 influenza A virus (IAV) infection. Overexpression of PD-L1 promoted viral replication and downregulated type-I and type-III interferons and interferon-stimulated genes. Moreover, the association between PD-L1 and Src homology region-2, containing protein tyrosine phosphatase (SHP2), during IAV/H1N1 infection was analyzed by employing the SHP2 inhibitor (SHP099), siSHP2, and pNL-SHP2. The results showed that the expressions of PD-L1 mRNA and protein were decreased under SHP099 or siSHP2 treatment, whereas the cells overexpressing SHP2 exhibited the opposite effects. Additionally, the effects of PD-L1 on the expression of p-ERK and p-SHP2 were investigated in PD-L1-overexpressed cells following WSN or PR8 infection, determining that the PD-L1 overexpression led to the decreased expression of p-SHP2 and p-ERK induced by WSN or PR8 infection. Taken together, these data reveal that PD-L1 could play an important role in immunosuppression during IAV/H1N1 infection; thus, it may serve as a promising therapeutic target for development of novel anti-IAV drugs.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Influenza, Human , Humans , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Influenza A Virus, H1N1 Subtype/metabolism , Influenza, Human/genetics , Influenza, Human/metabolism , Influenza A virus/physiologyABSTRACT
H9N2 is currently the main subtype of avian influenza in China. In order to use reverse genetics to rapid preparation of seed strains for vaccine production, and intend to prevent and control the H9N2 subtype epidemic strains of avian influenza virus (AIV). In this study, we successfully rescued 2 H9N2 recombinant viruses based on the representative viruses of Southeast China and confirmed by RT-PCR and sequencing. Genetic stability, pathogenicity, transmissibility, and antigenicity of 2 recombinant viruses were evaluated. Compared to the FZ1, the growth kinetics of H9N2(HA+NA)/PR8 showed no significant difference, H9N2(HA+NA+M+PB1)/PR8 was slightly lower. Our study also confirmed 2 recombinant viruses had good genetic stability after 10 passages but possessed lower pathogenicity than FZ1. Although both recombinant viruses led to seroconversion in all inoculated birds on 14 dpi, they complete loss of viral transmission of the virus to contact birds. In addition, birds were immunized via hypodermic route by inactivated vaccines of H9N2(HA+NA)/PR8, H9N2(HA+NA+M+PB1)/PR8 and wild-type virus with a single dose, and the results showed that the hemagglutination inhibition titers on 21 dpv were 10.5, 9.6, and 10.5 log2, respectively. And recombinant viruses both provided a certain protection against wild-type virus challenge. In conclusion, these data indicated that 2 recombinant viruses will be expected to be used as inactivated vaccines to controlling the spread of H9N2 subtype AIV even have potential application for attenuated viral vaccines, which provides a reference for the prevention and control of influenza virus pandemics.
Subject(s)
Influenza A Virus, H9N2 Subtype , Influenza Vaccines , Influenza in Birds , Animals , Influenza in Birds/prevention & control , Influenza A Virus, H9N2 Subtype/genetics , Chickens , Virulence , Vaccines, InactivatedABSTRACT
Circular RNAs (circRNAs) are an important subclass of noncoding RNAs implicated in the regulation of multiple biological processes. However, the functional involvement of circRNAs in the pathogenesis of influenza A viruses (IAVs) remains largely unknown. Here, we employed RNA sequencing (RNA-Seq) to examine the differentially expressed circRNAs in mouse lung tissues challenged or not challenged with IAV to evaluate the impact of viral infection on circRNAs in vivo. We observed that 413 circRNAs exhibited significantly altered levels following IAV infection. Among these, circMerTK, the derivative of myeloid-epithelial-reproductive tyrosine kinase (MerTK) pre-mRNA, was highly induced by IAV. Interestingly, circMerTK expression was also increased upon infection with multiple DNA and RNA viruses in human and animal cell lines, and thus it was selected for further studies. Poly(I:C) and interferon ß (IFN-ß) stimulated circMerTK expression, while RIG-I knockout and IFNAR1 knockout cell lines failed to elevate circMerTK levels after IAV infection, demonstrating that circMerTK is regulated by IFN signaling. Furthermore, circMerTK overexpression or silencing accelerated or impeded IAV and Sendai virus replication, respectively. Silencing circMerTK enhanced the production of type I IFNs and interferon-stimulating genes (ISGs), whereas circMerTK overexpression suppressed their expression at both the mRNA and protein levels. Notably, altering circMerTK expression had no effect on the MerTK mRNA level in cells infected or not infected with IAV, and vice versa. In addition, human circMerTK and mouse homologs functioned similarly in antiviral responses. Together, these results identify circMerTK as an enhancer of IAV replication through suppression of antiviral immunity. IMPORTANCE CircRNAs are an important class of noncoding RNAs characterized by a covalently closed circular structure. CircRNAs have been proven to impact numerous cellular processes, where they conduct specialized biological activities. In addition, circRNAs are believed to play a crucial role in regulating immune responses. Nevertheless, the functions of circRNAs in the innate immunity against IAV infection remain obscure. In this study, we employed transcriptomic analysis to investigate the alterations in circRNAs expression following IAV infection in vivo. It was found that expression of 413 circRNAs was significantly altered, of which 171 were upregulated, and 242 were downregulated following the IAV infection. Interestingly, circMerTK was identified as a positive regulator of IAV replication in both human and mouse hosts. CircMerTK was shown to influence IFN-ß production and its downstream signaling, enhancing IAV replication. This finding provides new insights into the critical roles of circRNAs in regulating antiviral immunity.
ABSTRACT
MIR155HG encodes a precursor RNA of microRNA-155 (miRNA-155). We previously identified this RNA also as a long noncoding RNA (lncRNA) that we call lncRNA-155. To define the functions of miRNA-155 and lncRNA-155, we generated miRNA-155 knockout (KO) mice lacking only 19 bp of the miRNA-155 core sequence without affecting the expression of lncRNA-155. Surprisingly, compared with the miRNA-155KO mice, previously generated lncRNA-155KO mice were more susceptible to both influenza virus (RNA virus) and pseudorabies virus (DNA virus) infection, as characterized by lower survival rate, higher body weight loss, and higher viral load. We found that miRNA-155-5p enhanced antiviral responses by positively regulating activation of signal transducer and activator of transcription 1 (STAT1), but the STAT1 activity differed greatly in the animals (lncRNA-155KO < miRNA-155KO < wild type). In line with this, expression levels of several critical interferon-stimulated genes (ISGs) were also significantly different (lncRNA-155KO < miRNA-155KO < wild type). We found that lncRNA-155 augmented interferon beta (IFN-ß) production during the viral infection, but miRNA-155 had no significant effect on the virus-induced IFN-ß expression. Furthermore, we observed that lncRNA-155 loss in mice resulted in dramatic inhibition of virus-induced activation of interferon regulatory factor 3 compared to both miRNA-155KO and wild-type (WT) animals. Moreover, lncRNA-155 still significantly suppressed the viral infection even though the miRNA-155 derived from lncRNA-155 was deleted or blocked. These results reveal that lncRNA-155 and miRNA-155 regulate antiviral responses through distinct mechanisms, indicating a bivalent role for MIR155HG in innate immunity. IMPORTANCE Here, we found that lncRNA-155KO mice lacking most of the lncRNA-155 sequences along with pre-miRNA-155, were more susceptible to influenza virus or pseudorabies virus infection than miRNA-155KO mice lacking only 19 bp of the miRNA-155 core sequence without affecting the expression of lncRNA-155, as evidenced by faster body weight loss, poorer survival, and higher viral load, suggesting an additional role of lncRNA-155 in regulating viral pathogenesis besides via processing miRNA-155. Congruously, miRNA-155-deleted lncRNA-155 significantly attenuated the viral infection. Mechanistically, we demonstrated miRNA-155-5p potentiated antiviral responses by promoting STAT1 activation but could not directly regulate the IFN-ß expression. In contrast, lncRNA-155 enhanced virus-induced IFN-ß production by regulating the activation of interferon regulatory factor 3. This finding reveals a bivalent role of MIR155HG in regulating antiviral responses through encoding lncRNA-155 and miRNA-155-5p and provides new insights into complicated mechanisms underlying interaction between virus and host innate immunity.
Subject(s)
MicroRNAs , RNA, Long Noncoding , Virus Diseases , Viruses , Animals , Mice , Antiviral Agents , RNA, Long Noncoding/genetics , Interferon Regulatory Factor-3/metabolism , Virus Replication/genetics , Immunity, Innate/genetics , Interferon-beta/genetics , MicroRNAs/genetics , Viruses/genetics , Weight LossABSTRACT
STAT2 is an important transcription factor activated by interferons (IFNs) upon viral infection and plays a key role in antiviral responses. Interestingly, here we found that phosphorylation of STAT2 could be induced by several viruses at early infection stage, including influenza A virus (IAV), and such initial activation of STAT2 was independent of type I IFNs and JAK kinases. Furthermore, it was observed that the early activation of STAT2 during viral infection was mainly regulated by the RIG-I/MAVS-dependent pathway. Disruption of STAT2 phosphorylation at Tyr690 restrained antiviral response, as silencing STAT2 or blocking STAT2 Y690 phosphorylation suppressed the expression of several interferon-stimulated genes (ISGs), thereby facilitating viral replication. In vitro experiments using overexpression system or kinase inhibitors showed that several kinases including MAPK12 and Syk were involved in regulation of the early phosphorylation of STAT2 triggered by IAV infection. Moreover, when MAPK12 kinase was inhibited, expression of several ISGs was clearly decreased in cells infected with IAV at the early infection stage. Accordingly, inhibition of MAPK12 accelerated the replication of influenza virus in host. These results provide a better understanding of how initial activation of STAT2 and the early antiviral responses are induced by the viral infection.
Subject(s)
Influenza A virus , Influenza, Human , Interferon Type I , Antiviral Agents/pharmacology , Humans , Interferon Type I/metabolism , Janus Kinases/metabolism , STAT2 Transcription Factor/metabolismABSTRACT
Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus type 2 (PCV2), has ravaged the pig industry, causing huge economic loss. At present, PCV2b and PCV2d are highly prevalent genotypes worldwide, while in China, in addition to PCV2b and PCV2d, a newly emerged PCV2e genotype detected in the Fujian province has attracted attention, indicating that PCV2 genotypes in China are more abundant. A preliminary study was conducted to better understand the genetic diversity and prevalence of PCV2 genotypes in southern China. We collected 79 random lung samples from pigs with respiratory signs, from 2018 to 2021. We found a PCV2-positivity rate of 29.1%, and frequent co-infections of PCV2 with PCV3, Streptococcus suis (S. suis), and other porcine pathogens. All PCV2-positive samples were sequenced and subjected to whole-genome analysis. Phylogenetic analysis, based on the PCV2 ORF2 gene and complete genomes, found that PCV2 strains identified in this study belonged to genotypes PCV2a (1), PCV2b (6), PCV2d (10), and PCV2e (6). Importantly, PCV2e was identified for the first time in some provinces, including Guangdong and Jiangxi. Additionally, we found two positively selected sites in the ORF2 region, located on the previously reported antigenic epitopes. Moreover, codon 63, one of the positively selected sites, has different types of amino acids in different genotypes. In conclusion, this study shows that PCV2e is an emerging genotype circulating in southern China, which warrants urgent, specific surveillance to aid the development of prevention and control strategies in China.
Subject(s)
Circoviridae Infections , Circovirus , Swine Diseases , Animals , China/epidemiology , Circoviridae Infections/epidemiology , Circoviridae Infections/veterinary , Circovirus/genetics , Genetic Variation , Genotype , Phylogeny , Swine , Swine Diseases/epidemiologyABSTRACT
The neuropeptide S (NPS) and its receptor (NPSR) represent a signaling system in the brain. Increased levels of NPS and NPSR have been observed in PK15 cells and murine brains in response to pseudorabies virus (PRV) infection, but it remains unclear whether elevated levels of NPS and NPSR are involved in the pathogenic process of PRV infection. In this study, the activities of both NPS and NPSR during PRV pathogenesis were explored in vitro and in vivo by reverse transcription polymerase chain reaction (RT-PCR), PCR, real-time quantitative RT-PCR (qRT-PCR), qPCR, TCID50, and Western blotting methods. NPSR-deficient cells were less susceptible to PRV infection, as evidenced by decreased viral production and PRV-glycoprotein E (gE) expression. In vitro studies showed that exogenous NPS promoted the expression of interleukin 6 (IL-6) mRNA but inhibited interferon ß (IFN-ß) mRNA expression in PK15 cells after PRV infection. In vivo studies showed that NPS-treated mice were highly susceptible to PRV infection, with decreased survival rates and body weights. In addition, NPS-treated mice showed elevated levels of IL-6 mRNA and STAT3 phosphorylation. However, the expression of IFN-ß mRNA was greatly decreased after virus challenge. Contrasting results were obtained from the NPSR-ir-treated groups, which further highlighted the effects of NPS. This study revealed that NPS-treated hosts are more susceptible to PRV infection than controls. Moreover, excessive IL-6/STAT3 and defective IFN-ß responses in NPS-treated mice may contribute to the pathogenesis of PRV.
Subject(s)
Herpesvirus 1, Suid , Neuropeptides , Pseudorabies , Rodent Diseases , Animals , Herpesvirus 1, Suid/genetics , Interleukin-6 , Mice , Neuropeptides/metabolism , RNA, MessengerABSTRACT
Spleen tyrosine kinase (Syk) has recently come forth as a critical regulator of innate immune response. Previous studies identify Syk as a key kinase for STAT1 activation at the early stage of influenza A virus (IAV) infection that is involved in initial antiviral immunity. However, the involvement of Syk in host antiviral immunity during the late phase of IAV infection and its effect on pathogenesis of the virus remain unknown. Here, we found through time course studies that Syk restrained antiviral immune response at the late stage of IAV infection, thereby promoting viral replication. Depletion of Syk suppressed IAV replication in vitro, whereas ectopic expression of Syk facilitated viral replication. Moreover, Syk-deficient mice were employed, and we observed that knockout of Syk rendered mice more resistant to IAV infection, as evidenced by a lower degree of lung injury, slower body weight loss, and an increased survival rate of Syk knockout mice challenged with IAV. Furthermore, we revealed that Syk repressed the interferon response at the late stage of viral infection. Loss of Syk potentiated the expression of type I and III interferons in both Syk-depleted cells and mice. Mechanistically, Syk interacted with TBK1 and modulated its phosphorylation status, thereby impeding TBK1 activation and restraining innate immune signaling that governs interferon response. Together, these findings unveil a role of Syk in temporally regulating host antiviral immunity and advance our understanding of complicated mechanisms underlying regulation of innate immunity against viral invasion. IMPORTANCE Innate immunity must be tightly controlled to eliminate invading pathogens while avoiding autoimmune or inflammatory diseases. Syk is essential for STAT1 activation at the early stage of IAV infection, which is critical for initial antiviral responses. Surprisingly, here a time course study showed that Syk suppressed innate immunity during late phases of IAV infection and thereby promoted IAV replication. Syk deficiency enhanced the expression of type I and III interferons, inhibited IAV replication, and rendered mice more resistant to IAV infection. Syk impaired innate immune signaling through impeding TBK1 activation. These data reveal that Syk participates in the initiation of antiviral defense against IAV infection and simultaneously contributes to the restriction of innate immunity at the late stage of viral infection, suggesting that Syk serves a dual function in regulating antiviral responses. This finding provides new insights into complicated mechanisms underlying interaction between virus and host immune system.
Subject(s)
Immunity, Innate , Influenza A virus , Orthomyxoviridae Infections , Animals , Antiviral Agents/metabolism , Host Microbial Interactions/immunology , Humans , Interferons/metabolism , Mice , Orthomyxoviridae Infections/enzymology , Orthomyxoviridae Infections/immunology , Syk Kinase/genetics , Syk Kinase/immunology , Virus ReplicationABSTRACT
African swine fever is one of the most devastating swine diseases caused by African swine fever virus (ASFV). Although ASFV encodes more than 160 viral proteins, the implication of a majority of ASFV proteins in regulating host immunity is yet to be explored, and the mechanisms of immune evasion by ASFV proteins are largely unknown. Here, we report that the I226R protein of ASFV significantly suppressed innate immune responses. The ectopic expression of ASFV I226R in 293T cells significantly inhibited the activation of interferon-stimulated response element promoters triggered by Sendai virus (SeV), poly(I:C), or cyclic GMP-AMP synthase (cGAS)/STING. The I226R protein caused a significant decrease in the expression of interferons and interferon-stimulating genes in cells infected with SeV. Similar results were obtained from experiments using I226R-overexpressed PK15 and 3D4/21 cells stimulated with vesicular stomatitis virus. We observed that I226R inhibited the activation of both nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). Furthermore, it was shown that overexpression of I226R suppressed IRF3 activation and caused the degradation of NF-κB essential modulator (NEMO) protein. The I226R-induced NEMO degradation could be prevented by treatment with MG132, a proteasome inhibitor. Together, these results reveal that the ASFV I226R protein impairs antiviral responses, likely through multiple mechanisms including the suppression of NF-κB and IRF3 activation, to counteract innate immune responses during the viral infection.
Subject(s)
African Swine Fever Virus , African Swine Fever , African Swine Fever Virus/physiology , Animals , Antiviral Agents/metabolism , Immunity, Innate , Interferons/metabolism , NF-kappa B/metabolism , Signal Transduction , SwineABSTRACT
BACKGROUND: Dysregulation of long noncoding RNAs (lncRNAs) has been linked to various human cancers. Bcr-Abl oncogene that results from a reciprocal translocation between human chromosome 9 and 22, is associated with several hematological malignancies. However, the role of lncRNAs in Bcr-Abl-induced leukemia remains largely unexplored. METHODS: LncRNA cDNA microarray was employed to identify key lncRNAs involved in Bcr-Abl-mediated cellular transformation. Abl-transformed cell survival and xenografted tumor growth in mice were evaluated to dissect the role of imatinib-upregulated lncRNA 1 (IUR1) in Abl-induced tumorigenesis. Primary bone marrow transformation and in vivo leukemia transplant using lncRNA-IUR1 knockout (KO) mice were further conducted to address the functional relevance of lncRNA-IUR1 in Abl-mediated leukemia. Transcriptome RNA-seq and Western blotting were performed to determine the mechanisms by which lncRNA-IUR1 regulates Bcr-Abl-induced tumorigenesis. RESULTS: We identified lncRNA-IUR1 as a critical negative regulator of Bcr-Abl-induced tumorigenesis. LncRNA-IUR1 expressed in a very low level in Bcr-Abl-positive cells from chronic myeloid leukemia patients. Interestingly, it was significantly induced in Abl-positive leukemic cells treated by imatinib. Depletion of lncRNA-IUR1 promoted survival of Abl-transformed human leukemic cells in experiments in vitro and xenografted tumor growth in mice, whereas ectopic expression of lncRNA-IUR1 sensitized the cells to apoptosis and suppressed tumor growth. In concert, silencing murine lncRNA-IUR1 in Abl-transformed cells accelerated cell survival and the development of leukemia in mice. Furthermore, lncRNA-IUR1 deficient mice were generated, and we observed that knockout of murine lncRNA-IUR1 facilitated Bcr-Abl-mediated primary bone marrow transformation. Moreover, animal leukemia model revealed that lncRNA-IUR1 deficiency promoted Abl-transformed cell survival and development of leukemia in mice. Mechanistically, we demonstrated that lncRNA-IUR1 suppressed Bcr-Abl-induced tumorigenesis through negatively regulating STAT5-mediated GATA3 expression. CONCLUSIONS: These findings unveil an inhibitory role of lncRNA-IUR1 in Abl-mediated cellular transformation, and provide new insights into molecular mechanisms underlying Abl-induced leukemogenesis.
Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Genes, abl , Imatinib Mesylate/pharmacology , Protein Kinase Inhibitors/pharmacology , RNA, Long Noncoding , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Disease Models, Animal , Fusion Proteins, bcr-abl/genetics , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Gene Expression Profiling , Humans , Imatinib Mesylate/therapeutic use , Mice, Knockout , Protein Kinase Inhibitors/therapeutic use , STAT5 Transcription Factor/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The nonreceptor tyrosine kinase ITK is a key component of the T cell receptor (TCR) signaling pathway and is required for cytokine production by CD4+ T cells that have differentiated into Th2 cells. Structural and biochemical studies suggest that contacts between the SH2 and SH3 domains of ITK mediate intermolecular self-association, forming a structure that restrains ITK activity by interfering with interactions between ITK and other components of the TCR signaling pathway. Wild-type (WT) ITK and a panel of ITK mutants containing amino acid substitutions in the SH2 and SH3 domains were tested for self-association and for binding to the adaptor protein SLP76, a key ligand for the ITK SH2 domain. WT and ITK mutants were also expressed in Itk-deficient CD4+ T cells via retroviral-mediated gene delivery to analyze their ability to support TCR signaling and cytokine production by Th2 cells. Specific amino acid substitutions in the ITK SH2 or SH3 domains impaired self-association, with the greatest effects being seen when both intermolecular SH2-SH3 domain contacts were disrupted. Two of the SH2 domain substitutions tested reduced ITK self-association but had no effect on binding to SLP-76. When their function was analyzed in Th2 cells, ITK proteins with diminished self-association activity supported greater IL-4 production and calcium flux in response to TCR stimulation compared to WT ITK. Our findings indicate that intermolecular contacts between ITK molecules can restrain the amplitude of TCR signaling, suggesting ITK is a limiting factor for responses by CD4+ T cells.
Subject(s)
Signal Transduction , src Homology Domains , CD4-Positive T-Lymphocytes/metabolism , Cytokines/metabolism , Protein Binding , Receptors, Antigen, T-Cell/genetics , Th2 CellsABSTRACT
Streptococcus suis serotype 2 (S. suis 2) is an important ubiquitous zoonotic pathogen. To date, regulatory factors and their implication in S. suis pathogenesis are not fully understood. Small non-coding RNAs (sRNAs) have been proven to function as important regulatory factors in bacterial pathogenesis and stress adaptation. Here, we identified a differentially downregulated S. suis 05ZYH33 sRNA after iron starvation by RNA-seq, which we named sRNA23. The presence of sRNA23 was further confirmed by RACE and Northern blot. Expression of sRNA23 was significantly altered under different environmental stresses such as nutritional starvation, osmotic pressure, oxidative stress, and lysozymal exposure. A sRNA23-deleted mutant exhibited relatively shorter streptococcal chains and weakened biofilm-forming ability. The mutation also resulted in decreased adherence of the S. suis 05ZYH33 to human laryngeal epidermoid carcinoma (HEp-2) cells, increased sensitivity to phagocytosis by RAW264.7 macrophages, and significantly reduced hemolytic activity. Furthermore, we observed that a sRNA23-deleted mutant had a low survival rate in pig whole blood and attenuated virulence in a mouse model. Moreover, based on RNA pull-down and electrophoretic mobility shift assay, we found that sRNA23 can directly bind to two proteins involved in adhesion and biofilm formation, namely, moonlighting protein FBA (fructose diphosphate aldolase) and rplB (50S ribosomal protein L2), respectively. Collectively, sRNA23 enhances S. suis 2 pathogenicity and the binding between sRNA23 and FBA/rplB might play an essential role in the adherence and biofilm-forming ability of S. suis 2.