Tumor suppressor p53 protects mice against Listeria monocytogenes infection.

Tumor suppressor p53 is involved in regulating immune responses, which contribute to antitumor and antiviral activity. However, whether p53 has anti-bacterial functions remains unclear. Listeria monocytogenes (LM) causes listeriosis in humans and animals, and it is a powerful model for studying innate and adaptive immunity. In the present study, we illustrate an important regulatory role of p53 during LM infection. p53 knockout (p53KO) mice were more susceptible to LM infection, which was manifested by a shorter survival time and lower survival rate. p53KO mice showed significant impairments in LM eradication. Knockdown of p53 in RAW264.7 and HeLa cells resulted in increased invasion and intracellular survival of LM. Furthermore, the invasion and intracellular survival of LM was inhibited in p53-overexpressing RAW264.7 and HeLa cells. LM-infected p53KO mice exhibited severe clinical symptoms and organ injury, presumably because of the abnormal production of the pro-inflammatory cytokines TNF-α, IL-6, IL-12, and IL-18. Decreased IFN-γ and GBP1 productions were observed in LM-infected p53-deficient mice or cells. The combination of these defects likely resulted in the overwhelming LM infection in the p53KO mice. These observations indicate that p53 serves as an important regulator of the host innate immune that protects against LM infection.

Transcriptional analysis of immune-related gene expression in p53-deficient mice with increased susceptibility to influenza A virus infection.

BACKGROUND: p53 is a tumor suppressor that contributes to the host immune response against viral infections in addition to its well-established protective role against cancer development. In response to influenza A virus (IAV) infection, p53 is activated and plays an essential role in inhibiting IAV replication. As a transcription factor, p53 regulates the expression of a range of downstream responsive genes either directly or indirectly in response to viral infection. We compared the expression profiles of immune-related genes between IAV-infected wild-type p53 (p53WT) and p53-deficient (p53KO) mice to gain an insight into the basis of p53-mediated antiviral response. METHODS: p53KO and p53WT mice were infected with influenza A/Puerto Rico/8/1934 (PR8) strain. Clinical symptoms and body weight changes were monitored daily. Lung specimens of IAV-infected mice were collected for analysis of virus titers and gene expression profiles. The difference in immune-related gene expression levels between IAV-infected p53KO and p53WT mice was comparatively determined using microarray analysis and confirmed by quantitative real-time reverse transcription polymerase chain reaction. RESULTS: p53KO mice showed an increased susceptibility to IAV infection compared to p53WT mice. Microarray analysis of gene expression profiles in the lungs of IAV-infected mice indicated that the increased susceptibility was associated with significantly changed expression levels in a range of immune-related genes in IAV-infected p53KO mice. A significantly attenuated expression of Ifng (encoding interferon (IFN)-gamma), Irf7 (encoding IFN regulator factor 7), and antiviral genes, such as Mx2 and Eif2ak2 (encoding PKR), were observed in IAV-infected p53KO mice, suggesting an impaired IFN-mediated immune response against IAV infection in the absence of p53. In addition, dysregulated expression levels of proinflammatory cytokines and chemokines, such as Ccl2 (encoding MCP-1), Cxcl9, Cxcl10 (encoding IP-10), and Tnf, were detected in IAV-infected p53KO mice during early IAV infection, reflecting an aberrant inflammatory response. CONCLUSION: Lack of p53 resulted in the impaired expression of genes involved in IFN signaling and the dysregulated expression of cytokine and chemokine genes in IAV-infected mice, suggesting an essential role of p53 in the regulation of antiviral and inflammatory responses during IAV infection.

Annexin 2 is a host protein binding to classical swine fever virus E2 glycoprotein and promoting viral growth in PK-15 cells.

Glycoprotein E2 of classical swine fever virus (CSFV) is a key determinant and major immunogen for viral entry and immunity, but little is known about its interaction with host proteins. In a previous study, we showed by proteomic analysis that cellular membrane protein annexin 2 (Anx2) was up-regulated in PK-15 cells following CSFV infection, but its function in CSFV replication remains unknown. In the present study we observed the interaction of Anx2 with CSFV E2 following infection of PK-15 cells by co-immunoprecipitation (Co-IP), mass spectrometry, Western blot and confocal laser scanning microscopy. The interaction between CSFV E2 and Anx2 was further confirmed in an E2-expressing PK-15 cell line, in which up-regulation of Anx2 was also observed, indicating that E2 alone can interact with, and increase, the expression of Anx2 protein. Further studies showed that siRNA-mediated knock-down and plasmid-mediated over-expression of Anx2 in PK-15 cells inhibited and increased CSFV replication and proliferation respectively. Remarkably, treatment of PK-15 cells with Anx2-specific polyclonal antibody prior to virus infection significantly inhibited CSFV multiplication, indicating that Anx2 is a cellular membrane protein likely associated with CSFV entry into cells. In conclusion, Anx2 is the novel host protein identified to interact with CSFV E2 and promote CSFV multiplication. These observations provide support for the potential use of Anx2 as a cellular target for the development of novel anti-CSFV therapies.

Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53.

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2'-5'-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.

Molecular characterization of enterohemorrhagic E. coli O157 isolated from animal fecal and food samples in Eastern China.

OBJECTIVE: To elucidate the extent of food contamination by enterohemorrhagic Escherichia coli (EHEC) O157 in Eastern China. METHODS: A total of 1100 food and animal fecal samples were screened for EHEC O157. Then, molecular characterization of each isolate was determined. RESULTS: EHEC O157 was isolated as follows: pig feces, 4% (20/500); cattle feces, 3.3% (2/60); chicken feces, 1.43% (2/140); pork, 2.14% (3/140), milk, 1.67% (1/60); and chicken meat, 1.67% (1/60). The stx1, stx2, eae, and hlyA genes were present in 26.7% (8/30), 40% (12/30), 63.3% (19/30), and 50% (15/30) of the O157 isolates, respectively. Molecular typing showed that strains from fecal and food samples were clustered into the same molecular typing group. Furthermore, the isolates from pork and pig feces possessed the same characterization as the clinical strains ATCC35150 and ATCC43889. Biofilm formation assays showed that 53.3% of the EHEC O157 isolates could produce biofilm. However, composite analyses showed that biofilm formation of EHEC O157 was independent of genetic background. CONCLUSIONS: Animal feces, especially from pigs, serve as reservoirs for food contamination by EHEC O157. Thus, it is important to control contamination by EHEC O157 on farms and in abattoirs to reduce the incidence of foodborne infections in humans.

Nitazoxanide inhibits the replication of Japanese encephalitis virus in cultured cells and in a mouse model.

BACKGROUND: Japanese encephalitis virus (JEV) has a significant impact on public health. An estimated three billion people in 'at-risk' regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing. Consequently, there is an urgent need for the development of novel therapeutic agents against Japanese encephalitis. Nitazoxanide (NTZ) is a thiazolide anti-infective licensed for the treatment of parasitic gastroenteritis. Recently, NTZ has been demonstrated to have antiviral properties. In this study, the anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model. METHODS: JEV-infected cells were treated with NTZ at different concentrations. The replication of JEV in the mock- and NTZ-treated cells was examined by virus titration. NTZ was administered at different time points of JEV infection to determine the stage at which NTZ affected JEV replication. Mice were infected with a lethal dose of JEV and intragastrically administered with NTZ from 1 day post-infection. The protective effect of NTZ on the JEV-infected mice was evaluated. FINDINGS: NTZ significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 ± 0.04 µg/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 ± 0.31 µg/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated by daily intragastric administration of 100 mg/kg/day of NTZ were protected from a lethal challenge dose of JEV. CONCLUSIONS: Both in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of Japanese encephalitis.

Identification of human guanylate-binding protein 1 gene (hGBP1) as a direct transcriptional target gene of p53.

Human guanylate-binding protein 1 (hGBP1) plays an important role in antitumor and antiviral immune responses. Here, we show that tumor suppressor p53 positively regulated hGBP1 transcription via binding to the p53 response element (p53RE) present in the hGBP1 promoter region. p53 activation by 5-fluorouracil significantly increased hGBP1 expression in wild-type p53 cells, but not in p53-null cells. Knockdown of p53 expression remarkably impaired hGBP1 expression induced by 5-fluorouracil, type I interferon treatment, or influenza A virus infection. Among three deductive p53REs present in the hGBP1 promoter region, two p53REs were found to be transactivated by p53.

Nonstructural protein 1 of influenza A virus interacts with human guanylate-binding protein 1 to antagonize antiviral activity.

Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1.

Detection and new genetic environment of the pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in methicillin-resistant Staphylococcus aureus of swine origin.

OBJECTIVES: To investigate the genetic basis of pleuromutilin resistance in porcine methicillin-resistant Staphylococcus aureus (MRSA) and to map the genetic environment of the identified plasmid-borne resistance gene. METHODS: Seventy porcine MRSA isolates, which exhibited high MICs of tiamulin, valnemulin and retapamulin, were investigated for pleuromutilin resistance genes and mutations. They were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). Plasmid DNA was extracted from the lsa(E)-positive strains and transferred to S. aureus RN4220 for selection of resistance plasmids. The plasmid-borne lsa(E) gene region was sequenced and 10 overlapping PCR assays for the analysis of the genetic environment of lsa(E) were developed. RESULTS: All 70 MRSA isolates were ST9 (MLST)-t899 (spa)-IVa (SCCmec). Sixteen isolates carried the lsa(E) gene; all others were negative for known pleuromutilin resistance mechanisms. An lsa(E)-carrying plasmid of ∼41 kb was detected in a single isolate. Sequence analysis revealed that the lsa(E) gene was located in a multiresistance gene cluster, which showed partial homology to clusters identified in MRSA, methicillin-susceptible S. aureus (MSSA) and Enterococcus faecalis. PCR analysis of the remaining isolates revealed a partly deleted multiresistance gene cluster in 6/15 isolates and solely the lsa(E) gene without the known flanking regions in 9/15 isolates. CONCLUSIONS: We identified the pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in porcine MRSA isolates. The multiresistance gene cluster in which lsa(E) was located differed from the previously described ones found in human MRSA/MSSA or in E. faecalis. The location of lsa(E) on a multiresistance plasmid facilitates its persistence and dissemination.

Down-regulation of cellular protein heme oxygenase 1 inhibits proliferation of classical swine fever virus in PK-15 cells.

Heme oxygenase 1 (HO-1) is an inducible enzyme that exerts potent antioxidant and anti-inflammatory effects, which also plays a critical role in host defenses against microbial, and particularly viral, infections. In our previous study, up-regulation of HO-1 was observed in peripheral blood leukocytes (PBLs) by genomic expression profiling, following infection of pigs with virulent classical swine fever virus (CSFV), the causative agent of a highly contagious disease threatening global pig industry (Shi et al., 2009). To study the potential involvement of HO-1 in CSFV proliferation, the role of its down-regulation in CSFV-infected PK-15 cells was further investigated. Results showed that infection with virulent CSFV strain Shimen significantly up-regulated the expression of HO-1 and that its down-regulation by small interfering RNA (siRNA) could inhibit CSFV proliferation as measured by genomic replication and production of infectious virus. The study revealed the involvement of HO-1 in CSFV proliferation, indicating that HO-1 is a potential target for inhibition of CSFV replication.

[Development of SYBR Green I real-time RT-PCR for the detection of Ebola virus].

In order to establish a rapid and accurate method for the detection of Ebola virus (EBOV), the primers used in SYBR Green I real-time RT-PCR were designed based on the EBOV NP gene sequences published in GenBank. The SYBR Green I real-time RT-PCR was established and optimized for the detection of EBOV. The EBOV RNA that was transcribed in vitro was used as a template. The sensitivity of this method was found to reach 1.0 x 10(2) copies/microL and the detection range was 10(2) - 10(10). No cross reaction with RNA samples from Marburg virus, Dengue virus, Xinjiang hemorrhagic fever virus, Japanese encephalitis virus, Influenza virus (H1N1 and H3N2) and Porcine reproductive and respiratory syndrome virus E genomic RNA was found. The method would be useful for the detection and monitoring of EBOV in China.

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

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.

[Advances in ubiquitin-like protein ISG15-mediated anti-viral response].

ISG15 is a 15kD ubiquitin-like protein (UBL) induced by interferon (IFN). ISG15 can be covalently attached to proteins, which is called ISGylation process. ISGylation system contains ISG15, UBE1L, UBCH8 and HERC5 proteins, which are all essential for ISGylation. ISG15 and ISGylation system have been found to have anti-viral effects. A better understanding of how ISG15 mediates the anti-viral activity will provide insights for new anti-viral drugs development and new therapeutic strategies. The mechanisms underlying the ISG15 mediated anti-viral response have been explored extensively in recent years. This minireview summarized the research advances of how ISG15 mediated the anti-viral effects against different kinds of viruses.

Generation and epitope mapping of a monoclonal antibody against nucleoprotein of Ebola virus.

Ebola virus (EBOV) causes highly lethal hemorrhagic fever in humans and nonhuman primates and has a significant impact on public health. The nucleoprotein (NP) of EBOV (EBOV-NP) plays a central role in virus replication and has been used as a target molecule for disease diagnosis. In this study, we generated a monoclonal antibody (MAb) against EBOV-NP and mapped the epitope motif required for recognition by the MAb. The MAb generated via immunization of mice with prokaryotically expressed recombinant NP of the Zaire Ebola virus (ZEBOV-NP) was specific to ZEBOV-NP and able to recognize ZEBOV-NP expressed in prokaryotic and eukaryotic cells. The MAb cross-reacted with the NP of the Reston Ebola virus (REBOV), the Cote-d'Ivoire Ebola virus (CIEBOV) and the Bundibugyo Ebola virus (BEBOV) but not with the NP of the Sudan Ebola virus (SEBOV) or the Marburg virus (MARV). The minimal epitope sequence required for recognition by the MAb was the motif PPLESD, which is located between amino acid residues 583 and 588 at the C-terminus of ZEBOV-NP and well conserved among all 16 strains of ZEBOV, CIEBOV and BEBOV deposited in GenBank. The epitope motif is conserved in four out of five strains of REBOV.

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-ß, 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.

Characterization of nonstructural protein 3 of a neurovirulent Japanese encephalitis virus strain isolated from a pig.

BACKGROUND: Japanese encephalitis virus (JEV), as a re-emerging virus that causes 10,000-15,000 human deaths from encephalitis in the world each year, has had a significant impact on public health. Pigs are the natural reservoirs of JEV and play an important role in the amplification, dispersal and epidemiology of JEV. The nonstructural protein 3 (NS3) of JEV possesses enzymatic activities of serine protease, helicase and nucleoside 5'-triphosphatase, and plays important roles in viral replication and pathogenesis. RESULTS: We characterized the NS3 protein of a neurovirulent strain of JEV (SH-JEV01) isolated from a field-infected pig. The NS3 gene of the JEV SH-JEV01 strain is 1857 bp in length and encodes protein of approximately 72 kDa with 99% amino acid sequence identity to that of the representative immunotype strain JaGAr 01. The NS3 protein was detectable 12 h post-infection in a mouse neuroblastoma cell line, Neuro-2a, and was distributed in the cytoplasm of cells infected with the SH-JEV01 strain of JEV. In the brain of mice infected with the SH-JEV01 strain of JEV, NS3 was detected in the cytoplasm of neuronal cells, including pyramidal neurons of the cerebrum, granule cells, small cells and Purkinje cells of the cerebellum. CONCLUSIONS: The NS3 protein of a neurovirulent strain of JEV isolated from a pig was characterized. It is an approximately 72 kDa protein and distributed in the cytoplasm of infected cells. The Purkinje cell of the cerebellum is one of the target cells of JEV infection. Our data should provide some basic information for the study of the role of NS3 in the pathogenesis of JEV and the immune response.

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.

Proteomic analysis of swine serum following highly virulent classical swine fever virus infection.

BACKGROUND: Classical swine fever virus (CSFV) belongs to the genus Pestivirus within the family Flaviviridae. Virulent strains of classical swine fever virus (CSFV) cause severe disease in pigs characterized by immunosuppression, thrombocytopenia and disseminated intravascular coagulation, which causes significant economic losses to the pig industry worldwide. METHODS: To reveal proteomic changes in swine serum during the acute stage of lethal CSFV infection, 5 of 10 pigs were inoculated with the virulent CSFV Shimen strain, the remainder serving as uninfected controls. A serum sample was taken at 3 days post-infection from each swine, at a stage when there were no clinical symptoms other than increased rectal temperatures (≥ 40 °C). The samples were treated to remove serum albumin and immunoglobulin (IgG), and then subjected to two-dimension differential gel electrophoresis. RESULTS: Quantitative intensity analysis revealed 17 protein spots showing at least 1.5-fold quantitative alteration in expression. Ten spots were successfully identified by MALDI-TOF MS or LTQ MS. Expression of 4 proteins was increased and 6 decreased in CSFV-infected pigs. Functions of these proteins included blood coagulation, anti-inflammatory activity and angiogenesis. CONCLUSION: These proteins with altered expression may have important implications in the pathogenesis of classical swine fever and provide a clue for identification of biomarkers for classical swine fever early diagnosis.

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

BACKGROUND: Marek's 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 Marek's 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. RESULTS: We 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. CONCLUSIONS: The 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.