| In eukaryotes, DExD/H helicase family has at least60members. Helicase is involved inunwinding double-stranded nucleic acids. Helicase is also involved in DNA repair andribosome biogenesis, transcriptional control of RNA metabolism. In recent years, more andmore RNA helicase family members have been recognized as important sensors in identifyingRNA or RNA viruses. The family is initially involved in RNA metabolic process. But later,we found that DHX9, DHX36and DDX41were able to identify the structure of DNA orDNA viruses. However, according to custom, the family is still named by RNA helicase.Double-stranded (dsRNA) is widely found in natural world, many ssRNA viruses alsoproduce dsRNAs during replication. Viral RNAs are recognized as foreign antigen and cantrigger host innate immune responses. On the other hand, part of helicase family membersfunction served as host innate immune system’s first line of defense, timely and effectivelyidentify foreign viral invasion by various adapter proteins mediate the immune responsesagainst viral infections and activate downstream signaling pathways, which lead to theinduction of type I interferon and pro-inflammatory cytokines. In this paper, we will focus onanti-virus immune mechanisms in mouse mDC.We use siRNA to knockdown (KD) all of the60members among DExD/H-box familyand screen out DHX33. The innate immune responses to both long and short polyI: Cstimulation were down regulated. It indicates that DHX33in mDC is an important sensor insensing dsRNA and RNA virus. To this end, we start the study of DHX33senses dsRNA andRNA viruses in D2SC mDC.1. Study of DHX33senses dsRNA and viral RNA in the D2SCWe establish the stable expression DHX33shRNA D2SC mDC cell lines by lentiviraltechnology. The scrambled shRNA treated D2SCs were used as negative control, while theD2SC cells with stable expression of IPS-I shRNA were used as positive control. Afterstimulation with long or short polyI: C, the cell supernantants were analyzed by ELISA todetect the production of type I interferons (IFN-α and IFN-β). In DHX33knockdown D2SC,both long and short polyI: C induced type I interferon production reduced significantlycompared with the control group. This result indicates that DHX33plays an important role in identifying both long and short polyI: C.Next we want to study whether DHX33senses the virus infection in D2SC mDC. Wecollect the supernatant of reovirus or LPS infected negative and positive control cells andDHX33KD D2SC mDC, use ELISA to test interferon response. In reovirus group, the IFNproduction in DHX33KD D2SC and IPS-1positive control group has significantly decreased.But after LPS stimulation, DHX33KD D2SC can hardly produce TNF-α and IL6. Thus, it isproved that DHX33can also recognize RNA viral infection in D2SC.2. Study of DHX33senses dsRNA and viral RNA in the BMDCsNext, we want to find whether DHX33sense dsRNA and viral RNA in primary cells. Weprepare BMDCs. The scrambled shRNA treated BMDCs served as negative control, while theBMDCs with stable expression of IPS-I shRNA served as positive control. After stimulationwith long or short polyI: C, the cell supernantants were analyzed by ELISA to detect theproduction of type I interferons (IFN-α and IFN-β). In DHX33knockdown BMDCs, bothlong and short polyI: C induced type I interferon production reduced significantly comparedwith the control group. This result indicates that DHX33plays an important role in identifyingboth long and short polyI: C in BMDCs.Next we want to study whether DHX33senses the virus infection in BMDCs. We collectthe supernatant of reovirus or LPS infected negative and positive control cells and DHX33KD BMDCs, using ELISA to detect interferon response. In reovirus group, the IFNproduction in DHX33KD BMDCs and IPS-1positive control group has significantlydecreased. But after LPS stimulation, DHX33KD BMDCs can hardly produce TNF-α andIL6. Thus, it is proved that DHX33can also recognize RNA viral infection in BMDCs.3．Restructuring DHX33”remedy” dsRNA immune response in DHX33knockdownD2SCWe add pCMV-HA vector and pCMV-HA-DHX33vector into DHX33knockdownD2SC and observe DHX33expression through IB. We find that, the DHX33expression inpCMV-HA vector group was significantly decreased compared with the control group, whilethe DHX33expression in the recombinant DHX33vector group get remedy. Further, we testthe IFN production using long polyI: C and short polyI: C to stimulate two groups of cells byELISA. It indicated that the interferon levels have been significantly restored in therecombinant DHX33vector group rather than in vector group.4. DHX33-dependent signaling pathwayIn DHX33knockdown and the IPS-1knockdown RIG-I deficient MEFs and DHX33 knockdown and the IPS-1knockdown MDA-5deficient MEFs, we use IB to test DHX33andIPS-1knockdown efficiency with IB assay. Then, using QPCR to reconfirm the target genesknockdown efficiency again. Finally, using short polyI: C to stimulate the MEFs, IFN levelswere examined by ELISA. Compared with the control group, we can see that the productionof IFNα still significantly decreased in both DHX33KD RIG-I deficient MEFs and MDA-5deficient MEFs. It proved that DHX33mediates signaling pathway are irrelevant with MDA5and RIG-I.5. Study of DHX33and polyI:C bindingWe use HA-tagged full-length DHX33and series of truncations were incubated withbiotin labeled polyI:C and pulldown with streptavidin beads. The relationship betweenHELICc DHX33domain and polyI:C has been proved through IP.To understand the specific binding of DHX33and polyI: C. We designed the competitionexperiments. The mixture of HA-DHX33with biotinylated polyI: C was added with anincremental gradient of unlabeled polyI: C or polyU, followed by neutral proteins avidinbeads pull down. We found that only unlabeled polyI: C can impede the binding ofbiotinylated polyI: C with DHX33, which further confirmed that the binding between DHX33and polyI: C is specific.6. Exploring the relationship between DHX33and IPS-1Myc-IPS-1and full-length HA-DHX33or series of truncations were incubated by anti-Myc beads pulldown. Contrarily, HA-DHX33and full-length Myc-IPS-1or series oftruncations were incubated with anti-HA beads pulldown. The combination between DHX33HELICc and IPS-I C-terminal region has been proved by IP individually.7. Study of downstream signaling pathway of DHX33and IPS-1Since IRF3and p65are dimeric forms of IFNs and NF-kB respectively. Erk1/2and p38are important molecules which participate in MAPK signal pathway. So, we start tounderstand the signaling pathway between DHX33and IPS-1by tracking thesephosphorylated molecules.Schramble shRNA control cells, IPS-1KD D2SC and DHX33KD D2SC werestimulated with long polyI:C. Whole cell protein lysates was extracted and separated bySDS-PAGE immunoblotting. In the control group, phophorylated p65, Erk1/2, p38and IRF3between time points in60minutes to120minutes could be detected. But, in IPS-1KD D2SCand DHX33KD D2SC, no protein bands were detected at any time point in phosphorylationof p65, Erk1/2, p38and IRF3. Thus it proved that the DHX33combined with the IPS-1 mediated signaling pathway is required for IRF3, NF-kB and MAPK activation.Through the above experiment, we obtained the following conclusions:1. In the D2SC mDC and BMDCs, DDX33can recognize both dsRNA and RNA viruses.2. Recombinant DHX33can rescue the immune response to dsRNA in DHX33KDD2SC.3. DHX33mediates signaling pathway is not dependent on RIG-I or MDA5.4. The binding between DHX33HELICc domain and polyI: C is specific.5. The HELICc domain of DHX33interacts with C-terminal region of IPS-1.6. DHX33mediates signaling pathway is required for MAP kinase, NF-kB and IRF3activation.Achievements creativities and status in research field:In helicase family, we find a new molecule DHX33that can recognize dsRNA and RNAvirus. Through further study, we find that DHX33mediates signaling pathway is notdependent on RIG-I or MDA5, indicating DHX33interacts with IPS-1adapter protein maymediate a new signaling pathway. DHX33is a potential target molecule in infectious diseasestreatment. |
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