Lilan Xie

Molecular cloning and functional characterization of porcine IFN-β promoter stimulator 1 (IPS-1)

The IFN-beta promoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for the retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that recognizes intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, porcine IPS-1 cDNA was cloned, using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR, from porcine peripheral blood mononuclear cells. The open reading frame of porcine IPS-1 consists of 1575bp encoding 524 amino acids. The putative porcine IPS-1 protein contains a N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts, ranging from 59% to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 produced type I IFN, and activated IRF3 and NF-kappaB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-beta promoter activation in PK-15 cells was significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in the porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in the type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.

Quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the JAK-STAT1 signaling pathway.

Transmissible gastroenteritis virus (TGEV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea and severe dehydration in piglets. In this study, liquid chromatography-tandem mass spectrometry coupled to isobaric tags for relative and absolute quantification labeling was used to quantitatively identify differentially expressed cellular proteins after TGEV infection in PK-15 cells. In total, 162 differentially expressed cellular proteins were identified, including 60 upregulated proteins and 102 downregulated proteins. These differentially expressed proteins were involved in the cell cycle, cellular growth and proliferation, the innate immune response, etc. Interestingly, many upregulated proteins were associated with interferon signaling, especially signal transducer and activator of transcription 1 (STAT1) and interferon-stimulated genes (ISGs). Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction demonstrated that TGEV infection induces STAT1 phosphorylation and nuclear translocation, as well as ISG expression. This study for the first time reveals that TGEV induces interferon signaling from the point of proteomic analysis.

Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

Abstract To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses.

Molecular cloning and functional characterization of porcine stimulator of interferon genes (STING).

The stimulator of interferon genes (STING) of human/mouse has been identified recently as an adaptor that links virus-sensing receptors to interferon regulatory factor 3 (IRF3) activation. Here we report the cloning and characterization of porcine STING (poSTING). The full-length poSTING cDNA sequence encodes 378 amino acids and contains one endoplasmic reticulum (ER) retention motif, RAR. Phylogenetic analysis revealed that poSTING, together with bovine STING, is more closely related to the human than to mouse STING. poSTING mRNA expression was mainly detected in the spleen, lymph node and lung. Enhanced green fluorescence protein (EGFP)-labeled poSTING was found to reside predominantly in the ER, and also in the mitochondrial membrane in PK-15 cells. Over-expression of poSTING activated both IRF3 and nuclear factor kappaB (NF-kappaB) transcription factors to induce IFN-beta production, while knockdown of poSTING significantly inhibited poly(I:C)- and poly(dAT:dAT)-induced IFN-beta promoter activation and IFN-beta mRNA production. Furthermore, the pseudorabies virus (PRV), a dsDNA virus, has been shown to activate the IFN-beta promoter in a poSTING-dependent way in porcine cell lines. Altogether, these results indicate that STING is an important regulator of porcine innate immune signaling. The results will help better understand the biological role(s) of STING in innate immunity during evolution.

Transmissible gastroenteritis virus infection induces NF-κB activation through RLR-mediated signaling

Abstract Transmissible gastroenteritis virus (TGEV) is a porcine enteric coronavirus which causes lethal severe watery diarrhea in piglets. The pathogenesis of TGEV is strongly associated with inflammation. In this study, we found that TGEV infection activates transcription factors NF-κB, IRF3 and AP-1 in a time- and dose-dependent manner in porcine kidney cells. Treatment with the NF-κB-specific inhibitor BAY11-7082 significantly decreased TGEV-induced proinflammatory cytokine production, but did not affect virus replication. Phosphorylation of NF-κB subunit p65 and proinflammatory cytokine production were greatly decreased after knockdown of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) or its adaptors MAVS and STING, while only slight reduction was observed in cells following silencing of Toll-like receptor adaptors, MyD88 and TRIF. Furthermore, TGEV infection significantly upregulated mRNA expression of RIG-I and MDA5. Taken together, our results indicate that the RLR signaling pathway is involved in TGEV-induced inflammatory responses.

Molecular cloning and functional characterization of porcine DNA-dependent activator of IFN-regulatory factors (DAI)

The DNA-dependent activator of IFN-regulatory factors (DAI) is a recently identified DNA sensor for intracellular DNA that triggers a signal for the production of type I IFN. Here we report the cloning and characterization of porcine DAI (poDAI). The full-length of poDAI encodes 439 amino acids, contains two N-terminal DNA-binding domains and shows similarity to mouse, rat, dog, monkey, human, horse and cattle counterparts ranging from 44% to 67%. poDAI mRNA expression was mainly detected in spleen, lung, kidney and small intestine. Over-expression of poDAI activated transcription factors IRF3 and NF-kappaB and induced IFN-beta in different porcine cell lines, but to varying degrees. Deletion mutant analysis revealed that both the DNA-binding domains and the C-terminus are required for full activation of IFN-beta. siRNA targeting poDAI significantly decreased poly(dAT:dAT)- or Pseudorabies virus (PRV)-induced IFN-beta activation. These results indicate that DAI is an important immuno-regulator of the porcine innate immune system.

Cellular RNA Helicase DDX1 Is Involved in Transmissible Gastroenteritis Virus nsp14-Induced Interferon-Beta Production

Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus (CoV) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. Unlike most CoVs that antagonize type I interferon (IFN) production, previous studies showed that TGEV infection induces IFN-I production both in vivo and in vitro. However, the underlying mechanism(s) remain largely unknown. In this study, we found that TGEV infection significantly facilitated IFN-β production as well as activation of the transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor-kappaB (NF-κB) in porcine kidney (PK-15) cells. Screening of TGEV-encoded proteins demonstrated that non-structural protein 14 (nsp14) was the most potent IFN-β inducer and induced IFN-β production mainly by activating NF-κB but not IRF3. Further analysis showed that nsp14 interacted with DDX1, a member of the DExD/H helicase family. Knockdown of DDX1 by specific small interfering RNA (siRNA) significantly decreased nsp14-induced IFN-β production and NF-κB activation. Furthermore, TGEV-induced IFN-β production and IFN-stimulated gene (ISG) expression were decreased in cells transfected with DDX1-specific siRNA, indicating the vital role of DDX1 to TGEV-induced IFN-β responses. In summary, our data revealed a potential coactivator role of host RNA helicase DDX1 to the induction of IFN-β response initiated by TGEV and demonstrated that nsp14 is an important IFN inducer among the TGEV-encoded proteins.

Porcine bocavirus NP1 protein suppresses type I IFN production by interfering with IRF3 DNA-binding activity.

Type I interferon (IFN) and the IFN-induced cellular antiviral responses are the primary defense mechanisms against viral infection; however, viruses always evolve various mechanisms to antagonize this host’s IFN responses. Porcine bocavirus (PBoV) is a newly identified porcine parvovirus. In this study, we found that the nonstructural protein NP1 of PBoV inhibits Sendai virus-induced IFN-β production and the subsequent expression of IFN-stimulating genes (ISGs). Ectopic expression of NP1 significantly impairs IRF3-mediated IFN-β production; however, it does not affect the expression, phosphorylation, and nuclear translocation of IRF3, the most important transcription factor for IFN synthesis. Coimmunoprecipitation and Chromatin immunoprecipitation assays suggested that NP1 interacts with the DNA-binding domain of IRF3, which in turn blocks the association of IRF3 with IFN-β promoter. Together, our findings demonstrated that PBoV encodes an antagonist inhibiting type I IFN production, providing a better understanding of the PBoV immune evasion strategy.