The Mechanism Studies Of DDX3 In Regulating NF-?B Signaling Pathway

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 et al.Recent studies revealed that DDX3 participates in innate immune response during virus infection,in which it interacts with TBK1 and regulates the production of IFN-?.In our studies,we demonstrated that DDX3 also regulate NF-?B signal pathway and revealed its regulatory mechanisms.We also found that DDX3 can regulate the replication of PRRSV and the mechanisms were also been revealed simply.The main contents of this thesis are the following:1.DDX3 can affect the production of inflammatory cytokinesThe cytokine production is one of the important indicators of the activation of the innate immune responses.In order to study whether DDX3 affected the production of the proinflammatory cytokines,we detected the production of several inflammatory cytokines using qRT-PCR after the expression of DDX3 in HeLa cells was silenced with small interfering RNA(siRNA)specific targeting DDX3(siDDX3).The qRT-PCR results showed that the mRNA levels of of IL-1,IL-6,IL-8 and TNF-a were decreased whether stimulated by poly(I:C)or TNF-a.These results suggested that DDX3 affected the production of proinflammatory cytokines.2.DDX3 participates in the regulation of NF-?B signal pathwayIt is well-known that cytokine production can be regulated by the NF-?B signal pathway.Meanwhile,both poly(I:C)and TNF-a could activate NF-?B signal pathway.Therefore,we speculated that DDX3 might regulate inflammatory cytokine production through NF-?B signal pathway.Western blot analysis showed that the phosphorylation of p65 in HeLa cells stimulated by poly(I:C)or TNF-a was decreased after DDX3 expression was knocked down.P65 is released after I?B?phosphorylation and degradation and translocates into the nucleus to activate the transcription of multiple target genes.The phosphorylation and degradation of I?B?are associated with the phosphorylation of p65.Our data revealed that the phosphorylation and degradation of IKBa were attenuated in DDX3 knockdown cells.The catalytic activity of IKK-? contributes essentially to I?B? phosphorylation and NF-?B activation.Our results also confirmed that DDX3 knockdown reduced the phosphorylation of the upstream signaling molecule IKK-?.We also found that the nuclear translocation of p65 was reduced after DDX3 knockdown.All these results suggested that DDX3 regulated the NF-?B signal pathway.3.DDX3 regulates the interaction between IKK-? and PP2A-CIn order to study the mechanism on how DDX3 regulates the NF-?B signal pathway,we used the Flag-DDX3 as the bait to perform the immunoprecipitation experiment.The immunoprecipitated proteins were further analyzed by mass spectrometry.In the list,we found the candidate protein,the catalytic subunit of the protein phosphatase 2A(PP2A-C),a major serine/threonine phosphatase in eukaryotic cells.We next studied the possible interaction between DDX3 and PP2A-C subunit by co-immunoprecipitation using DDX3 and PP2A-C over-expressing cells,which confirmed that the Flag-tagged DDX3 interacted with HA-tagged PP2A-C subunit.We also confirmed that DDX3 and PP2A-C subunit can interact with each other in physiological condition by immunoprecipitation of the endogenous proteins.More in-depth studies have shown that PP2A-C interacted with IKK-? and the interaction between PP2A-C and IKK-p was enhanced after DDX3 knockdown.On the other hand,overexpression of IKK-? mutant(S177/181E)restored the decreasing tendency of phosphorylation of p65 which caused by knockdown of DDX3.All this means that DDX3 regulates the interaction between IKK-? and PP2A-C,which augments the dephosphorylation of IKK-? by PP2A-C.4.DDX3 controls the phosphorylation of PP2A-CThe phosphorylation state of PP2A-C could affect the catalytic activity of PP2A and consequently influence the NF-?B signal pathway.So we wondered whether DDX3 might regulate the NF-?B signal pathway by controlling the phosphorylation of PP2A-C.Our western blot analysis showed that DDX3 knockdown reduced the phosphorylation of PP2A-C at Tyr307.The non-functional mutant PP2A-C(Y307D)is the phosphorylation-mimicking mutation that abolished the phosphatase activity of PP2 A.Overexpression of this mutant increased the phosphorylation of IKK-p and p65.PP2A-C mutant(Y307D)restored the decreased phosphorylation of p65 caused by DDX3 knockdown.These results indicated that DDX3 regulated NF-?B signal pathway by controlling the phosphorylation of PP2A-C.5.A preliminary study about the effect of DDX3 on PRRSV replicationDDX3 is involved in the regulation of innate immune responses,especially in regulating the production of interferon.Many viruses exploit or inhibit DDX3 proteins to complete their replication.We have tentatively studied the effect of DDX3 on PRRSV replication.The results showed that DDX3 act as a double-edged sword in PRRSV replication.When DDX3 is absent,replication of PRRSV is reduced.But DDX3 is the necessary protein to produce interferon.DDX3 knockdown reduced the phosphorylation of IRF3.In-depth studies,we found that DDX3 is ubiquitinated for degradation during interferon activation.DDX3 ubiquitination mutants(K335R,K342R)enhanced the phosphorylation of IRF3 and reduced the replication of PRRSV.It means that the replication of PRRSV requires DDX3 and PRRSV can inhibit the production of interferon by regulating the ubiquitination of DDX3.Further studies showed that DDX3 interacts with the NSP9 protein of PRRSV.The interaction between DDX3 and NSP9 may play an important role in PRRSV replication.