Yafeng Qiu

Influenza A virus induces p53 accumulation in a biphasic pattern.

Tumor suppressor p53, the major cellular defense against tumor development, has recently been implicated in host antiviral defense. Previous studies have shown that p53 was induced at the apoptotic stage of influenza virus-infected cells. However, we found that p53 was induced not only at the apoptotic stage but also at the beginning phase of infection, showing a biphasic pattern with a first transient elevation apparent at the beginning phase of infection and a second elevation observable at the middle-late phase of infection. This up-regulation of p53 was independent of increased p53 transcription, but dependent on virus adsorption and replication. The increased p53 was active and able to transactivate its downstream target genes, such as interferon regulatory factor 9 (IRF9) and Bax. To our knowledge, this is the first report to describe a biphasic pattern of p53 accumulation in influenza virus-infected cells.

The non-structural (NS1) protein of influenza A virus associates with p53 and inhibits p53-mediated transcriptional activity and apoptosis

NS1 protein of influenza A virus is involved in regulating the apoptosis of infected cells. We found that exogenously expressed NS1 was able to associate with the tumor suppressor p53 that plays an essential role in regulating apoptosis of influenza A virus-infected cells. Exogenous expression of NS1 resulted in inhibition of p53-mediated transcriptional activity and apoptosis. The p53 inhibitory domain of NS1 was located between amino acids 144 and 188. This domain is necessary for NS1 to inhibit p53 activity, but it requires additional region(s) to cooperatively exert this inhibitory function.

The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities

BackgroundMareks disease virus (MDV) is an oncogenic herpesvirus, which causes malignant lymphoma in chickens. The Meq protein of MDV, which is expressed abundantly in MDV-infected cells and in Mareks disease (MD) tumor cells, functions as a transcriptional activator and has been proposed to play an important role in oncogenic transformation. Preliminary studies demonstrated that Meq is able to bind p53 in vitro, as demonstrated using a protein-binding assay. This observation prompted us to examine whether the interaction between Meq and p53 occurs in cells, and to investigate the biological significance of this interaction.ResultsWe confirmed first that Meq interacted directly with p53 using a yeast two-hybrid assay and an immunoprecipitation assay, and we investigated the biological significance of this interaction subsequently. Exogenous expression of Meq resulted in the inhibition of p53-mediated transcriptional activity and apoptosis, as analyzed using a p53 luciferase reporter assay and a TUNEL assay. The inhibitory effect of Meq on transcriptional activity mediated by p53 was dependent on the physical interaction between these two proteins, because a Meq deletion mutant that lacked the p53-binding region lost the ability to inhibit p53-mediated transcriptional activity and apoptosis. The Meq variants L-Meq and S-Meq, but not VS-Meq and ∆Meq, which were expressed in MD tumor cells and MDV-infected cells, exerted an inhibitory effect on p53 transcriptional activity. In addition, ∆Meq was found to act as a negative regulator of Meq.ConclusionsThe Meq oncoprotein interacts directly with p53 and inhibits p53-mediated transcriptional activity and apoptosis. These findings provide valuable insight into the molecular basis for the function of Meq in MDV oncogenesis.

Stabilization of p53 in influenza A virus-infected cells is associated with compromised MDM2-mediated ubiquitination of p53.

Backgound: p53 is accumulated and activated in response to influenza virus infection. Results: p53 accumulation results from protein stabilization, which is associated with compromised Mdm2-mediated p53 ubiquitination. Viral nucleoprotein binds to p53 and impairs Mdm2-mediated p53 ubiquitination. Conclusion: p53 stabilization results from compromised Mdm2-mediated p53 ubiquitination. Significance: First time to demonstrate the mechanism of p53 stabilization in influenza virus-infected cells. Influenza A virus (IAV) induces apoptosis of infected cells. In response to IAV infection, p53, a tumor suppressor involved in regulating apoptosis and host antiviral defense, accumulates and becomes activated. This study was undertaken to examine the mechanism of p53 accumulation in IAV-infected cells. Here we show that p53 accumulation in IAV-infected cells results from protein stabilization, which was associated with compromised Mdm2-mediated ubiquitination of p53. In IAV-infected cells, p53 was stabilized and its half-life was remarkably extended. The ladders of polyubiquitinated p53 were not detectable in the presence of the proteasome inhibitor MG132 and were less sensitive to proteasome-mediated degradation. IAV infection did not affect the abundance of Mdm2, a major ubiquitin E3 ligase responsible for regulating p53 ubiquitination and degradation, but weakened the interaction between p53 and Mdm2. Viral nucleoprotein (NP) was able to increase the transcriptional activity and stability of p53. Furthermore, NP was found to associate with p53 and to impair the p53-Mdm2 interaction and Mdm2-mediated p53 ubiquitination, demonstrating its role in inhibiting Mdm2-mediated p53 ubiquitination and degradation.

Polyclonal antibody to porcine p53 protein: a new tool for studying the p53 pathway in a porcine model.

Although the tumor suppressor protein p53 is important in the control of various cellular activities, the analysis of p53 in the porcine model has been hampered by a lack of a suitable antibody that is specific for porcine p53. Using a recombinant porcine p53, we generated a rabbit polyclonal antibody (designated SH0797) that is directed against porcine p53. The results of the study show that the antibody is capable of detecting recombinant p53 protein expressed in Escherichia coli, as well as FLAG-tagged p53 that is expressed in the transfected cells. This demonstrates that the antibody is specific for the porcine p53 protein. The antibody also showed the ability to immunoprecipitate p53 protein from extracts of porcine cells and to cross-react with human p53 protein. In addition, expression of porcine p53 could be induced readily in porcine cells and detected using this new tool. This antibody is a useful tool for use in studies of the cellular pathways that involve p53 in the porcine model.

An hsp70 fusion protein vaccine potentiates the immune response against Japanese encephalitis virus

Summary.To evaluate the possibility of developing an effective subunit vaccine against Japanese encephalitis virus (JEV), mice were intraperitoneally immunized with either a neutralizing epitope (a 27-amino-acid region of the JEV E protein), or with a fusion protein between this region and a Mycobacterium tuberculosis hsp70. Both antigens were heterologously expressed in Escherichia coli as fusion proteins with thioredoxin. The fusion protein antigen elicited a higher titer of anti-thioredoxin-neutralizing epitope antibodies and a stronger proliferation of lymphocytes than did either the neutralizing epitope (irrespective of the presence of mineral oil as an adjuvant), or the conventional JEV SA14-14-2 vaccine. Assays of antibody isotype and IFN-γ and IL-4 content in post-immunization serum showed that the fusion protein elicited a higher IgG2a titer and higher levels of IFN-γ suggesting a potentiation of the Th1 immune response. The fusion protein antigen elicited a long-lived immune response, and the antibodies were able to neutralize JEV in vitro more strongly than did those elicited by the JEV SA14-14-2 vaccine. Immunization with the fusion protein generated both humoral and cellular immune responses to JEV, and the fusion protein appeared to be a more efficient protectant than the JEV SA14-14-2 vaccine.

Characterization of a novel small plasmid carrying the florfenicol resistance gene floR in Haemophilus parasuis

Sir, The Gram-negative bacterium Haemophilus parasuis is the causative agent of Glässer’s disease, which is characterized by various combinations of meningoencephalitis, polyserositis, polyarthritis and bacterial pneumonia, resulting in major economic losses to the swine industry worldwide. Florfenicol is used exclusively in veterinary medicine and was approved for the treatment of porcine respiratory diseases in China in 1999. So far, five florfenicol resistance genes (floR, fexA, fexB, cfr and optrA) have been reported in bacteria of animal origin. In Gram-negative bacteria, the floR gene is the most important contributor to florfenicol resistance and has been described in various species. To date, studies on the antimicrobial susceptibility of H. parasuis have been carried out in several countries (China, Denmark, the UK, Spain, the Czech Republic and Australia), which reported that all of the clinical isolates were susceptible to florfenicol. Here, we report the emergence of florfenicol-resistant H. parasuis isolates in China, which is attributable to a novel small plasmid bearing the floR gene. A total of 62 H. parasuis isolates were collected from pigs with respiratory diseases in Shanghai and Jiangsu province, China, from March 2013 to May 2014. Conventional biochemical testing, diagnostic PCR analysis and 16S rDNA sequencing were used to identify the isolates. Since there is no standard method for susceptibility testing of H. parasuis, broth microdilution was performed according to the recommendations of the CLSI (VET01-A4, 2013) for Actinobacillus pleuropneumoniae. A. pleuropneumoniae ATCC 27090 served as the quality control strain. Of the 62 H. parasuis isolates, 3 (ASB6W, ASB17W and A4) showed high florfenicol MIC values of 8 mg/L, while the remaining 59 strains exhibited low MIC values of florfenicol, ranging from 0.25 to 1 mg/L. The isolates ASB6W and ASB17W were obtained from a farm in Jiangsu province and the A4 isolate was from a farm in Shanghai. The three isolates were tentatively considered as florfenicol resistant according to the distribution of MIC values of the 62 H. parasuis isolates and the CLSI breakpoint of florfenicol for A. pleuropneumoniae. The presence of the floR gene in the three strains was confirmed by PCR with the primers floRF (5′-GCGATATTCATTACTTTGGC-3′) and floRR (5′-TAGGATGAAGG TGAGGAATG-3′) and subsequent sequencing of the amplicon. Plasmid DNA was extracted using the Qiagen Plasmid Mini Kit (Qiagen, Hilden, Germany) and profiling analysis revealed that each of the three florfenicol-resistant H. parasuis isolates harboured only a single plasmid of 6 kb. The aforementioned PCR fragment of the floR gene was labelled using the DIG High Prime DNA Labelling and Detection Kit (Roche Diagnostics, Mannheim, Germany). Southern blot analysis performed with the floR-specific probe showed that the floR gene was located on the 6 kb plasmids in the three isolates (data not shown). Subsequently, the three plasmids from the three isolates were completely sequenced by primer walking, starting with the floR-specific primers described above. Sequence comparisons revealed that the three plasmids were identical. This plasmid, designated as pHPSF1, was transformed into the plasmid-free H. parasuis isolate D20 by electrotransformation. The transformant exhibited elevated MICs of florfenicol (0.5–8 mg/L) and chloramphenicol (0.5–8 mg/L) when compared with those of the recipient strain. These results suggested that the plasmid pHPSF1 carried the floR gene and was responsible for florfenicol resistance in the three H. parasuis isolates. The plasmid pHPSF1 was 6328 bp (GenBank accession number KR262062) and consisted of five ORFs encoding the florfenicol resistance protein FloR, which consists of 404 amino acids, the 109 amino acid putative transcriptional regulator LysR, a potential Rep protein of 337 amino acids involved in plasmid replication and two Mob proteins associated with plasmid mobilization (MobC of 97 amino acids and MobA/L of 450 amino acids). The FloR protein of plasmid pHPSF1 had a typical size of 404 amino acids and 12 putative transmembrane domains, which is same as other functionally active members of the FloR family. It showed amino acid identity of 88.4% (to the FloR variant of Stenotrophomonas maltophilia, accession number AIU94575) to 97.5% (to the FloR of Escherichia coli, accession number CEL26452) with the FloR proteins in the GenBank database. Database searches identified three plasmids that showed high sequence homology with pHPSF1, including the 5486 bp plasmid p11745 carrying the tet(B) tetracycline resistance gene (GenBank accession number DQ176855) of A. pleuropneumoniae isolated from a pig in Spain, the 10 874 bp floR-carrying plasmid

Molecular cloning and functional characterization of a novel isoform of chicken myeloid differentiation factor 88 (MyD88)

Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor- and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). A novel isoform of chicken MyD88, designated chicken MyD88-2, has been cloned and functionally characterized. Its open reading frame is of length 900bp, and it encodes a predicted 299 residue protein, similar in length to its mammalian orthologues, but, respectively, 77 and 69 amino acids shorter than the previously described chicken MyD88-1 and -3. The amino acid sequence of chicken MyD88-2 displays 96.9%, 96.9%, 70.4% and 70.2% identity with, respectively, chicken MyD88-1, -3, human and mouse MyD88. Chicken MyD88-2 expression was detected in a range of tissues tested, but no expression of either chicken MyD88-1 or -3 was observed. The over-expression of chicken MyD88-2 significantly induced the activation of NF-kappaB in vitro, suggesting that chicken MyD88-2 plays an important role in the innate immune responses of chicken.

Icariin induces the Expression of Toll‐like Receptor 9 in Ana‐1 Murine Macrophages

Icariin is the major pharmacologically active compound of Herba epimedii which has been used as a tonic, aphrodisiac and an antirheumatic in traditional Chinese medicine. This study analysed the effect of icariin on the expression of Toll‐like receptor 9 (TLR9) which plays an important role in regulation of the innate immune response. Stimulation of Ana‐1 murine macrophages with icariin induced a significant dose‐dependent expression of TLR9, and its mRNA expression which increased from 3 h post‐treatment was approximately five‐fold that of DMSO‐treated cells. Several molecules, such as myeloid differentiation factor 88, tumor necrosis factor‐α and interleukin 6, which are involved in the TLR9 downstream signaling pathway, were also significantly up‐regulated in response to icariin stimulation. Our findings demonstrated that icariin is able to induce the expression of TLR9. Copyright

Glycyrrhetinic acid extracted from Glycyrrhiza uralensis Fisch. induces the expression of Toll-like receptor 4 in Ana-1 murine macrophages

Glycyrrhetinic acid (GA) is an active component of licorice root that has long been used as a herbal medicine for the treatment of peptic ulcer, hepatitis, and pulmonary and skin diseases in Asia and Europe. In this study, we analyzed the effect of GA extracted from Glycyrrhiza uralensis Fisch. on the expression of Toll-like receptors (TLRs) that play key roles in regulating the innate immune response against invading pathogens. Stimulation of Ana-1 murine macrophages with GA induced a significant dose-dependent expression of TLR-4, and its mRNA expression that increased from 3-h post-treatment was approximately fivefold over the level in the mock-treated cells. No endotoxin contamination contributed to the GA-induced TLR-4 expression, because polymyxin B treatment did not alter the upregulated expression of TLR-4 in GA-treated cells. Several molecules, such as myeloid differentiation factor 88, interferon-β, and interleukin-6, which are involved in the TLR-4 downstream signaling pathway, were upregulated significantly in response to GA stimulation. Our findings demonstrate that GA is able to induce the expression of TLR-4 and activate its downstream signaling pathway.