Identification of the internal ribosome entry sites in the 5'­untranslated region of the c­fos gene.

The Fos proto­oncogene, activator protein­1 (AP­1) transcription factor subunit (c­fos) gene, a member of the immediate early gene family, encodes c­Fos, which is a subunit of the AP­1 transcription factor. The present study aimed to investigate the mechanism by which the translation efficiency of c­fos mRNA is upregulated when cellular protein synthesis is shut off. The result of western blotting revealed that the protein expression levels of c­Fos were increased in rhabdomyosarcoma cells infected with enterovirus 71 (EV71) compared with uninfected cells. PCR was used to get the c­fos 5'­untranslated region (UTR). The luciferase assay of a bicistronic vector containing the c­fos 5'UTR revealed that the c­fos 5'UTR contains an internal ribosome entry site (IRES) sequence and a 175 nucleotide sequence (between 31 and 205 nt) that is essential for IRES activity. Analysis of potential IRES trans­acting factors revealed that poly(C)­binding protein 2 (PCBP2) negatively regulated the activity of the c­fos IRES, whereas the La autoantigen (La) positively regulated its activity. The results of RNA­protein immunoprecipitation demonstrated that both PCBP2 and La bound to the c­fos 5'UTR. Furthermore, the IRES activity of in vitro­transcribed c­fos mRNA was upregulated during EV71 infection. The present study suggested a mechanism for the effect of viral infection on host genes, and provided a novel target for gene translation regulation.

Ginsenoside Rb1 is an immune-stimulatory agent with antiviral activity against enterovirus 71.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine, the main pathogenesis of severe hand, foot and mouth disease (HFMD) is that the heat and wet poisons are deeply trapped in the viscera, which causes the deficiency of Qi and Yin in the patient's body. Ginsenoside Rb1 (Rb1) is the most abundant triterpenoid saponin in Panax quinquefolius L., which has the function of Qi-invigorating and Yin-nourishing. Enterovirus 71 (EV71) is one of the causative pathogens of HFMD, especially the form associated with some lethal complications. Therefore, the therapeutic effect of Rb1 on this disease caused by EV71 infection is worth exploring. AIM OF THE STUDY: We explored the effective antiviral activities of Rb1 against EV71 in vitro and in vivo and investigated its preliminary antiviral mechanisms. MATERIAL AND METHODS: EV71-infected two-day-old suckling mice model was employed to detect the antiviral effects of Rb1 in vivo. To detect the antiviral effects of Rb1 in vitro, cytopathic effect (CPE) reduction assay was performed in EV71-infected Rhabdomyosarcoma (RD) cells. Interferon (IFN)-ß interference experiment was employed to detect the antiviral mechanism of Rb1. RESULTS: In this paper, we first found that Rb1 exhibited strong antiviral activities in EV71-infected suckling mice when compared to those of ribavirin. Administration of Rb1 reduced the CPE of EV71-infected RD cells in a dose-dependent manner. Moreover, EV71-induced viral protein-1 (VP-1) expression was significantly reduced by Rb1 administration in vitro and in vivo. Furthermore, Rb1 treatment could induce high cellular and humoral immune responses in vivo. Meanwhile, Rb1 contributed to the enhanced Type I IFN responses and IFN-ß knockdown reversed the antiviral activity of Rb1 in vitro. CONCLUSION: In summary, our findings suggest that Rb1 is an immune-stimulatory agent and provide an insight into therapeutic potentials of Rb1 for the treatment of EV71 infection.

Neuropathogenicity of newly isolated avian leukosis viruses from chickens with osteopetrosis and mesenchymal neoplasms.

ABSTRACT The prototype fowl glioma-inducing virus (FGVp) causes fowl glioma and cerebellar hypoplasia in chickens. In this study, we investigated whether a strain of avian leukosis virus (ALV), associated with avian osteopetrosis and mesenchymal neoplasms, is able to induce fowl glioma. We encountered avian osteopetrosis and mesenchymal neoplasms, including myxosarcoma and rhabdomyosarcoma, in Japanese native chickens used for both egg-laying and meat production. These birds were also affected by non-suppurative encephalitis and glioma in their brains. Four ALV strains (GifN_001, GifN_002, GifN_004, GifN_005) were isolated, and a phylogenic analysis of envSU showed that these isolates were classified into different clusters from FGVp and the variants previously reported. Whereas the envSU shared a high identity (94.7%) with that of Rous sarcoma virus (strain Schmidt-Ruppin B) (RSV-SRB), the identity between envTM of GifN_001 and that of FGVp was high (94.5%), indicating that GifN_strains may emerge by recombination between FGVp and other exogenous ALVs. Specific-pathogen-free chickens inoculated in ovo with GifN_001 revealed fowl glioma and cerebellar hypoplasia. These results suggest that the newly isolated strains have acquired neuropathogenicity to chickens.

Exosomes cloak the virion to transmit Enterovirus 71 non-lytically.

Enterovirus 71 (EV71) is a non-enveloped virus and it can be released from host cells through a traditional cytolytic manner. Now, we showed EV71 could be spread non-lytically between cells during early viral infection. In order to explain this phenomenon, we separated supernatant fluids of rhabdomyosarcoma (RD) cells cultures infected with EV71 by isopycnic gradient centrifugation. Two populations of virus particles were morphology indistinguishable by transmission electron microscope (TEM). It showed that some EV71 particles were wrapped inside extracellular vesicles which were verified to be exosomes by immunoassay and morphologic analysis. In addition, exosomes containing viral RNA were shed in plasma of EV71-infected encephalitis in children. Our findings indicate that the "non-enveloped" EV71 virions could be wrapped within exosomes which promote their spread in the absence of cell lysis.Abbreviation: EV71: enterovirus 71; EXO: exosome; RD: rhabdomyosarcoma; TEM: transmission electron microscope; HFMD: hand, foot, and mouth disease; HIV: immunodeficiency virus; HCV: hepatitis C virus; HTLV: Human T-cell lymphotropic virus; HAV: hepatitis A virus; MOI: multiplicity of infection; EVs: extracellular vesicles; VP1: viral capsid protein 1; NTA: nanoparticle tracking analysis; CNS: central nervous system.

Enterovirus 71 Represses Interleukin Enhancer-Binding Factor 2 Production and Nucleus Translocation to Antagonize ILF2 Antiviral Effects.

Enterovirus 71 (EV71) infection causes hand-foot-mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children, thereby presenting a serious risk to public health. It is important to determine the mechanisms underlying the regulation of EV71 infection. In this study, we initially show that the interleukin enhancer-binding factor 2 (ILF2) reduces EV71 50% tissue culture infective dose (TCID50) and attenuates EV71 plaque-formation unit (PFU), thereby repressing EV71 infection. Microarray data analyses show that ILF2 mRNA is reduced upon EV71 infection. Cellular studies indicate that EV71 infection represses ILF2 mRNA expression and protein production in human leukemic monocytes (THP-1) -differentiated macrophages and human rhabdomyosarcoma (RD) cells. In addition, EV71 nonstructural protein 2B interacts with ILF2 in human embryonic kidney (HEK293T) cells. Interestingly, in the presence of EV71 2B, ILF2 is translocated from the nucleus to the cytoplasm, and it colocalizes with 2B in the cytoplasm. Therefore, we present a distinct mechanism by which EV71 antagonizes ILF2-mediated antiviral effects by inhibiting ILF2 expression and promoting ILF2 translocation from the nucleus to the cytoplasm through its 2B protein.

Large-Scale Proteomic Identification of Targets of Cellular miR-197 Downregulated by Enterovirus A71.

MicroRNAs are noncoding RNA species comprising 18-23 nucleotides that regulate host-virus interaction networks. Here, we show that enterovirus A71 infection in human rhabdomyosarcoma (RD) is regulated by miR-197 expression. Transfection of miR-197 mimic into RD cells inhibited virus replication by interfering with the viral RNA synthesis. We employed a combination of mass-spectrometry-based quantitative proteomics with the stable isotope labeling with amino acids in cell culture (SILAC) approach for the identification of the miR-197 target genes in RD cells and to investigate the differential expression of the prospective target proteins. A total of 1822 proteins were repeatedly identified in miR-197-transfected RD cells, 106 of which were predicted to have seed sites by TargetScan. Notably, seven of eight selected genes potentially related to viral replication and immune response were validated as direct miR-197 targets, using a luciferase 3'-untranslated region (UTR) reporter assay. The expression levels of three selected endogenous molecules (ITGAV, ETF1, and MAP2K1/MEK1) were significantly reduced when RD cells were transfected with a miR-197 mimic. Our results provide a comprehensive database of miR-197 targets, which might provide better insights into the understanding of host-virus interaction.

Characterization of Critical Functions of Long Non-Coding RNAs and mRNAs in Rhabdomyosarcoma Cells and Mouse Skeletal Muscle Infected by Enterovirus 71 Using RNA-Seq.

Enterovirus 71 (EV71) is the main pathogen of severe hand-foot-mouth disease (HFMD). Long non-coding RNAs (lncRNAs) are recognized as pivotal factors during the pathogenesis of viral infection. However, the critical functions of lncRNAs in EV71⁻host interactions have not been characterized. Here, for the first time, we performed global transcriptome analysis of lncRNA and mRNA expression profiles in EV71-infected human rhabdomyosarcoma (RD) cells and skeletal muscle of mice using second-generation sequencing. In our study, a total of 3801 novel lncRNAs were identified. In addition, 23 lncRNAs and 372 mRNAs exhibited remarkable differences in expression levels between infected and uninfected RD cells, while 104 lncRNAs and 2647 mRNAs were differentially expressed in infected skeletal muscle from neonatal mice. Comprehensive bioinformatics analysis included target gene prediction, lncRNA­mRNA co-expression network construction, as well as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis mainly focused on differentially-expressed genes (DEGs). Our results suggest that lncRNAs may participate in EV71 infection-induced pathogenesis through regulating immune responses, protein binding, cellular component biogenesis and metabolism. The present study provides novel insights into the functions of lncRNAs and the possible pathogenic mechanism following EV71 infection.

Antiviral activity of shikonin ester derivative PMM-034 against enterovirus 71 in vitro.

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants and children below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activities derived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviral activity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 on RD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cells were determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions were determined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibited significant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 µg/mL. The VP1 mRNA and protein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1ß, IL-6, IL-8, and TNF-α significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression was also significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.

Antiviral activity of shikonin ester derivative PMM-034 against enterovirus 71 in vitro

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants and children below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activities derived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviral activity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 on RD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cells were determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions were determined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibited significant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 μg/mL. The VP1 mRNA and protein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1β, IL-6, IL-8, and TNF-α significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression was also significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.

Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication.

Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.

The Transcriptome of Rhabdomyosarcoma Cells Infected with Cytolytic and Non-Cytolytic Variants of Coxsackievirus B2 Ohio-1.

The transcriptomes of cells infected with lytic and non-lytic variants of coxsackievirus B2 Ohio-1 (CVB2O) were analyzed using next generation sequencing. This approach was selected with the purpose of elucidating the effects of lytic and non-lytic viruses on host cell transcription. Total RNA was extracted from infected cells and sequenced. The resulting reads were subsequently mapped against the human and CVB2O genomes. The amount of intracellular RNA was measured, indicating lower proportions of human RNA in the cells infected with the lytic virus compared to the non-lytic virus after 48 hours. This may be explained by reduced activity of the cellular transcription/translation machinery in lytic enteroviral replication due to activities of the enteroviral proteases 2A and/or 3C. Furthermore, differential expression in the cells infected with the two virus variants was identified and a number of transcripts were singled out as possible answers to the question of how the viruses interact with the host cells, resulting in lytic or non-lytic infections.

The long non-coding RNA expression profile of Coxsackievirus A16 infected RD cells identified by RNA-seq.

Coxsackievirus A16 (CVA16) is one of major pathogens of hand, foot and mouth disease (HFMD) in children. Long non-coding RNAs (IncRNAs) have been implicated in various biological processes, but they have not been associated with CVA16 infection. In this study, we comprehensively characterized the landscape of IncRNAs of normal and CVA16 infected rhabdomyosarcoma (RD) cells using RNA-Seq to investigate the functional relevance of IncRNAs. We showed that a total of 760 IncRNAs were upregulated and 1210 IncRNAs were downregulated. Out of these dysregulated IncRNAs, 43.64% were intergenic, 22.31% were sense, 15.89% were intronic, 8.67% were bidirectional, 5.59% were antisense, 3.85% were sRNA host IncRNAs and 0.05% were enhancer. Six dysregulated IncRNAs were validated by quantitative PCR assays and the secondary structures of these IncRNAs were projected. Moreover, we conducted a bioinformatics analysis of an IncRNAs (ENST00000602478) to elucidate the diversity of modification and functions of IncRNAs. In summary, the current study compared the dysregulated IncRNAs profile upon CVA16 challenge and illustrated the intricate relationship between coding and IncRNAs transcripts. These results may not only provide a complete picture of transcription in CVA16 infected cells but also provide novel molecular targets for treatments of HFMD.

Gan-Lu-Siao-Du-yin, a prescription of traditional Chinese medicine, inhibited enterovirus 71 replication, translation, and virus-induced cell apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gan-Lu-Siao-Du-yin (GLSDY) is a prescription of traditional Chinese medicine. GLSDY contains 11 ingredients and is commonly used for endemic diseases. Enterovirus 71 (EV71) is an endemic disease that can cause meningoencephalitis with mortality and neurologic sequelae without any effective management. It is unknown whether GLSDY is effective against EV71 infection. AIM OF THE STUDY: To test the hypothesis that GLSDY can protect cell from EV71-induced injury. MATERIALS AND METHODS: Effects of a hot water extract of GLSDY on EV71 were tested in human foreskin fibroblast cells (CCFS-1/KMC) and human rhabdomyosarcoma cells (RD cells) by plaque reduction assay and flow cytometry respectively. Inhibition of viral replication was further examined by reverse quantitative RT-PCR (qRT-PCR). Its effect on viral protein translation and virus-induced apoptosis were examined by western blot. RESULTS: GLSDY was dose-dependently effective against EV71 infection (p<0.0001) in both CCFS-1/KMC cells and RD cells. GLSDY was highly effective when supplemented after viral inoculation (P<0.0001) with an IC50 of 8.7µg/mL. GLSDY inhibited viral RNA replication (P<0.0001), formation of viral structural proteins (VP0, VP1, VP2 and VP3) and non-structural proteins (protease 2B and 3AB). Furthermore, 300µg/mL GLSDY is effective to inhibit virus-induced apoptosis possibly through direct inhibition of caspase-8 and indirectly by inhibition of Bax. CONCLUSIONS: GLSDY is cheap and readily available to manage EV71 infection by inhibiting viral replication, viral protein formations, and EV71-induced apoptosis.

Oblongifolin M, an active compound isolated from a Chinese medical herb Garcinia oblongifolia, potently inhibits enterovirus 71 reproduction through downregulation of ERp57.

There is no effective drug to treat EV71 infection yet. Traditional Chinese herbs are great resources for novel antiviral compounds. Here we showed that Oblongifolin M (OM), an active compound isolated from Garcinia oblongifolia, potently inhibited EV71 infection in a dose dependent manner. To identify its potential effectors in the host cells, we successfully identified 18 proteins from 52 differentially expressed spots by comparative proteomics studies. Further studies showed that knockdown of ERp57 inhibited viral replication through downregulating viral IRES (internal ribosome entry site) activities, whereas ectopic expression of ERp57 increased IRES activity and partly rescued the inhibitory effects of OM on viral replication. We demonstrated that OM is an effective antiviral agent; and that ERp57 is one of its cellular effectors against EV71 infection.

[MiR432* regulate the replication of coxsackievirus A16 in rhabdomyosarcoma cells].

OBJECTIVE: MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells. In this study, we examined miRNAs' effects on the replication of Coxsackievirus A16 (CA16) in rhabdomyosarcoma cells. METHODS: We constructed target gene of miRNAs screening system. We used 3'untranslated region (UTR) dual luciferase reporter analysis to identify putative miRNA targets in the CA16 virus genome. First, 12 segments of CA16 virus genome were inserted to the pMIR vector and the luciferase expression were assayed to identify the target gene of putative miRNA. The reporter gene expression of the cells transfected with the vector containing 5'-UTR was significantly downregulated. Then, using online analysis programs we screened the miRNAs that may target to 5'-UTR. Furthermore, Western blot and real-time PCR test were used to study the effect of miRNAs on viral replication. RESULTS: The study showed that miR432 * could stimulate the replication of CA16 virus. On the contrary, miR432 * inhibitor could suppress CA16 virus replication. CONCLUSION: Cellular miRNAs could regulate the replication of CA16 virus in host cells. Our findings support the notion that the cellular miRNAs play an important role in the host and virus infection.

[MiR373 and miR542-5p regulate the replication of enterovirus 71 in rhabdomyosarcoma cells].

MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells. To identify cellular miRNAs involved in the host response to enterovirus 71 (EV71) infection, we examined miRNAs effects on the replication of EV71 in rhabdomyosarcoma cells. We constructed target gene of miRNAs screening system. 3'untranslated region (UTR) dual luciferase reporter analysis was used to identify putative miRNA targets in the EV71 virus genome. First, 13 segments of EV71 virus genomes were inserted to the pMIR vector and the luciferase expression were assayed to identify the target gene of putative miRNA. The reporter gene expression of the cells transfected with the vector containing 5'-UTR was significantly downregulated. Then we screened the miRNAs that may target to 5'-UTR using online analysis programs. Furthermore, Western blotting and real-time PCR test were performed to investigate the effect of miRNAs on viral replication. The study showed that miR373 and miR542-5p could suppress the replication of EV71 virus through binding to the 5'-UTR gene. Cellular miRNAs could regulate the replication of EV71 virus in host cells, and our paper should report the role of miR373 and miR542-5p in this regulation for the first time. Our findings supported the notion that the cellular miRNAs might be essential in the host-pathogen interactions.

[Pentapeptides prevent enterovirus 71 proliferation in rhabdomyosarcoma cells and mice].

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD). This article presented the inhibitory activity of pentapeptides on the EV71 infection in rhabdomyosarcoma (RD) and suckling mice. The EV71 VP1 capsid protein expression levels and mRNA levels were analyzed by Western blotting and real-time PCR. The antiviral activity of pentapeptides in vivo was evaluated by weight changes and EV71 VP1 protein expression levels in intestines of suckling mice. Results revealed that the pentapeptide P010157 was able to inhibit EV71 replication in RD cells. After being incubated with the P010157 at a concentration of 100 microg x mL(-1) for 48 h, the level of EV71 vp1 mRNA in RD cells decreased by (92.0 +/- 6.3)%. The estimated EC50 was 2.2 microg x mL(-1). P010157 was able to inhibit EV 71-induced cytopathic effect (CPE) in RD cells. The cytotoxic activity of the compound was evaluated against RD cells by MTS assay. The results showed that P010157 had no obvious toxicity. In addition, the treated mice with P010157 did not exhibit weight loss, as was observed in untreated mice. EV71 replication reduced significantly as revealed by Western blotting. These findings suggest that P010157 could prevent EV71 proliferation in vitro and in vivo. P010157 is a novel compound for antiviral therapies against EV71, which merited further investigation.

Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness.

Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-ß) gene (Ifn-ß) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Differential gene expressions of the MAPK signaling pathway in enterovirus 71-infected rhabdomyosarcoma cells

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells. METHODS: Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8 h and 20 h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA. RESULTS: The viability of RD cells decreased obviously within 48 h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p < 0.05). At 8 h after infection, the expressions of c-Jun, c-Fos, IFN-i, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08-6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20 h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased. CONCLUSION: EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.

Validation-based insertional mutagenesis for identification of Nup214 as a host factor for EV71 replication in RD cells.

Lentiviral validation-based insertional mutagenesis (VBIM) is a sophisticated, forward genetic approach that is used for the investigation of signal transduction in mammalian cells. Using VBIM, we conducted function-based genetic screening for host genes that affect enterovirus 71 (EV71) viral replication. This included host factors that are required for the life cycle of EV71 and host restriction factors that inhibit EV71 replication. Several cell clones, resistant to EV71, were produced using EV71 infection as a selection pressure and the nuclear pore protein 214 (Nup214) was identified as a host factor required for EV71 replication. In SD2-2, the corresponding VBIM lentivirus transformed clone, the expression of endogenous Nup214 was significantly down-regulated by the reverse inserted VBIM promoter. After Cre recombinase-mediated excision of the VBIM promoter, the expression of Nup214 recovered and the clone regained sensitivity to the EV71 infection. Furthermore, over-expression of Nup214 in the cells suggested that Nup214 was promoting EV71 replication. Results of this study indicate that a successful mutagenesis strategy has been established for screening host genes related to viral replication.