(DEAD)-box RNA helicase 3 modulates NF-κB signal pathway by controlling the phosphorylation of PP2A-C subunit.

Asp-Glu-Ala-Asp (DEAD)-box RNA helicase 3 (DDX3), an ATP-dependent RNA helicase, is associated with RNA splicing, mRNA export, transcription, translation, and RNA decay. Recent studies revealed that DDX3 participates in innate immune response during virus infection by interacting with TBK1 and regulating the production of IFN-ß. In our studies, we demonstrated that DDX3 regulated NF-κB signal pathway. We found that DDX3 knockdown reduced the phosphorylation of p65 and IKK-ß and ultimately attenuated the production of inflammatory cytokines induced by poly(I:C) or TNF-α stimulation. The regulatory effect of DDX3 on NF-κB signal pathway was not affected by the loss of its ATPase or helicase activity. We further identified PP2A C subunit (PP2A-C) as an interaction partner of DDX3 by co-immunoprecipitation and mass spectrum analysis. We confirmed that DDX3 formed the complex with PP2A-C/IKK-ß and regulated the interaction between IKK-ß and PP2A-C. Furthermore, we demonstrated that DDX3 modulated the activity of PP2A by controlling the phosphorylation of PP2A-C, which might enable PP2A-C to regulate NF-κB signal pathway by dephosphorylating IKK-ß. All these findings suggested DDX3 plays multiple roles in modulating innate immune system.

Glycyrrhizin inhibits porcine epidemic diarrhea virus infection and attenuates the proinflammatory responses by inhibition of high mobility group box-1 protein.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV) infection, leads to significant economic losses in the swine industry worldwide. In our studies, we found that glycyrrhizin, the major component of licorice root extracts, could moderately inhibit PEDV infection in Vero cells, when analyzed by western blot, qRT-PCR and a plaque formation assay. We also revealed that glycyrrhizin inhibited the entry and replication of PEDV. In addition, we demonstrated that glycyrrhizin decreased the mRNA levels of proinflammatory cytokines. Since glycyrrhizin is a competitive inhibitor of high mobility group box-1 (HMGB1), we confirmed that TLR4 and RAGE (£ associated with PEDV pathogenesis during the infection in Vero cells. In summary, our studies provide a molecular basis for developing novel therapeutic methods to control PEDV infection, based on glycyrrhizin and its derivatives.

Porcine epidemic diarrhea virus nucleoprotein contributes to HMGB1 transcription and release by interacting with C/EBP-ß.

Porcine epidemic diarrhea is a devastating swine enteric disease, which is caused by porcine epidemic diarrhea virus (PEDV) infection. Our studies demonstrated that PEDV infection resulted in the up-regulation of proinflammatory cytokines. Meanwhile, PEDV infection and overexpression of viral nucleoprotein resulted in the acetylation and release of high mobility group box 1 proteins in vitro, an important proinflammatory response mediator, which contributes to the pathogenesis of various inflammatory diseases. Our studies also showed that SIRT1, histone acetyltransferase, and NF-κB regulated the acetylation and release of HMGB1. Chromatin immunoprecipitation, dual-luciferase reporter gene assay, and co-immunoprecipitation experiments illustrated that PEDV-N could induce HMGB1 transcription by interacting with C/EBP-ß, which could bind to C/EBP motif in HMGB1 promotor region. Collectively, our data indicate PEDV-N contributes to HMGB1 transcription and the subsequent release/acetylation of HMGB1 during PEDV infection.

Syndecan-4, a PRRSV attachment factor, mediates PRRSV entry through its interaction with EGFR.

The causative agent of porcine reproductive and respiratory syndrome is the PRRS virus (PRRSV), an enveloped, single-stranded and positive-sense RNA virus. The host factors and mechanisms that are involved in PRRSV entry are still largely unknown. In our present studies, we found that syndecan-4, one of the heparan sulfate proteoglycans, plays a critical role in PRRSV entry, especially in PRRSV attachment. Moreover, EGFR interacts with syndecan-4 in MACR-145 cells and disruption of their interaction impaired PRRSV entry. Furthermore, EGFR inhibitor AG1478 or syndecan-4 derived peptide SSTN87-131 inhibited syndecan-4 endocytosis induced by PRRSV entry. Altogether, syndecan-4, a PRRSV attachment factor, mediated PRRSV entry by interacting with EGFR.

Porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor.

It is well known that many viruses use heparan sulfate as the initial attachment factor. In the present study, we determined whether porcine epidemic diarrhea virus (PEDV), an emerging veterinary virus, infects Vero cells by attaching to heparan sulfate. Western blot analysis, real-time PCR, and plaque formation assay revealed that PEDV infection was inhibited when the virus was pretreated with heparin (an analogue of heparan sulfate). There was no inhibitory effect when the cells were pre-incubated with heparin. We next demonstrated that enzymatic removal of the highly sulfated domain of heparan sulfate by heparinase I treatment inhibited PEDV infection. We also confirmed that sodium chlorate, which interferes with heparan sulfate biosynthesis, also inhibited PEDV infection. Furthermore, we examined the effect of two heparin derivatives with different types of sulfation on PEDV infection. The data suggested de-N-sulfated heparin, but not N-acetyl-de-O-sulfated heparin, inhibits PEDV infection. In summary, our studies revealed that heparan sulfate acts as the attachment factor of PEDV in Vero cells.

The involvement of FAK-PI3K-AKT-Rac1 pathway in porcine reproductive and respiratory syndrome virus entry.

CD163 and sialoadhesin had been reported as the two receptors for porcine reproductive and respiratory syndrome virus (PRRSV) infection. The signaling pathway activated by PRRSV entry was seldom reported. In our studies, we demonstrated that PRRSV entry triggers FAK, PI3K, AKT and Rac1 activation. The signaling pathway FAK-PI3K-AKT-Rac1 is essential for PRRSV entry. Blocking FAK by PF573228 attenuates the activation of PI3K, AKT, Rac1 and the cytoskeleton remodeling induced by virus entry. Inhibitors to FAK, PI3K, AKT and Rac1 can significantly inhibit the virus entry. In conclusion, our observations reveal that PRRSV triggers the activation of FAK-PI3K-AKT-Rac1 signaling pathway to facilitate its entry into cells.