Preliminary Study On The Role Of DDX17 In The Replication Of Hantaan Virus

Research BackgroundHantavirus is widely spread around the world,and it is mainly transmitted by rodents,whose infection can cause two acute infectious diseases,namely hemorrhagic fever with renal syndrome（HFRS）and Hantavirus pulmonary syndrome（HPS）.To note,most cases of HFRS are reported in China.Hantaan virus（HTNV）,the prototype of hantavirus,is the main causative agent of severe HFRS.HTNV belongs to the single negative strand RNA viruses（-ssRNA）,and its genome includes three segments,namely L,M and S,which encode RNA-dependent RNA polymerase（RdRp）,glycoprotein prescursor（GPC）and nucleocapsid protein（NP）,respectively.To date,there is no specific treatment for HFRS as the pathogenesis of HTNV infection still remains unclear.Therefore,in-depth study of the mechanism of HTNV infection and replication can provide a theoretical basis for HFRS pathogenesis,and provide potential targets for the development of novel therapeutic drugs.Virus-host interaction is a hot topic in the field of HTNV replication research in recent years.HTNV NP is abundantly expressed in the cytoplasm in the early stage of virus infection and plays an important role in the life cycle of the virus.NP promotes viral replication through interaction with viral components and host proteins.However,it is unclear how host proteins interact with NP,and its molecular mechanism as well as the biological function.Study on the interaction between HTNV NP and host proteins is of great significance for elucidating the mechanism of HTNV infection and replication.Research purposesIdentification of host proteins interacting with HTNV NP,and investigation of the function of DDX17 during HTNV infection.Method and result1.Identification of host proteins that interact with HTNV NP1.1 Construction of eukaryotic expression plasmids containing SF-NPFirst of all,the cDNA of S gene from HTNV（76-118 strain）was amplified by PCR with the primers including two restriction enzyme sites called EcoRⅠand KpnⅠin the upstream and downstream.Secondly,the synthetic gene fragments of Strep-Flag（SF）digested by two kinds of restriction enzymes,KpnⅠand XhoⅠ,were ligated with the pCAGGS plasmids that were digested by the same restriction enzymes,and formed the pCAGGS-SF.Thirdly,the NP fragments digested by EcoRⅠand KpnⅠwere inserted in the plasmid pCAGGS-SF,the recombinant plasmid named by pCAGGS-SF-NP.Finally,we transfected pCAGGS-SF-NP into HEK293T cells and showed that SF-NP could express correctly and effectively in eukaryote cell through Western blot.1.2 Isolation and identification of the host proteins interacting with NP by affinity chromatographyHEK293T cells were transfected with pCAGGS-SF-NP and pCAGGS-SF,respectively.Cell lysates were collected after 48 hours post transfection.Then the samples were incubated with StrepTrap^TM HP agar beads in a chromatography column at 4°C for12 hours.The host protein interacting with SF-NP was obtained after the step of desulfurization biotin elution,and subjected to SDS-PAGE and coomassie blue staining.Compared with the pCAGGS-SF transfection group,the pCAGGS-SF-NP transfection group showed specific bands at 75-60 kDa,35-25 kDa positions,named as No.1,No.2and No.3 respectively.The bands were cut down and sent to The Beijing Genomics Institute for identification through liquid chromatography.The results showed that DDX3,DDX17 and DDX5,which belong to the helicase family molecules and are widely involved in the viral life cycle,were found in the first band.Based on our previous study,DDX17 was selected as the candidate molecule for further study.2.Investigation of the role of DDX17 during HTNV infection2.1 Detection of the expression level and subcellular location of DDX17 after HTNV infectionHuman umbilical vein endothelial cells（HUVEC）were infected with HTNV at an MOI of 1 and the uninfected virus group was used as control.Cell lysates were harvested at 0 h,12 h,24 h,3 d and 5 d,and RNA were extracted from these cell samples.Then,the cDNA was prepared by RNA reverse transcription,and the expression level of DDX17mRNA at various time points was detected by real-time quantitative PCR（RT-PCR）.The results showed that the expression level of mRNA increased gradually and it reached the highest level at 24 hpi.Then it began to decrease,and returned to normal level on the 3 d,suggesting that DDX17 may play a role at the early stage of HTNV infection.The subcellular localization of DDX17 was detected by immunofluorescence assays at 24 hpi.The results indicated that DDX17 was mainly located in the nucleus of uninfected group,while DDX17 accumulated in cytoplasm after HTNV infection.2.2 The effect of DDX17 overexpression on HTNV replicationThe recombinant lentivirus containing pLVX-ZsGreen-DDX17 was constructed and packaged,after which the lentivirus titer was detected and the overexpression efficiency was confirmed.After DDX17 overexpression in HUVEC through lentivirus infection,we infected these cells with HTNV（MOI=1）.The cell lysates were harvested at 24 h,48 h,and 72 h.The replication of HTNV was assessed by RT-PCR,Western blot and in-cell western（ICW）.The results showed that the expression level of mRNA and structure protein of HTNV were significantly increased in DDX17 overexpression group,indicating that DDX17 can promote the replication of HTNV.2.3 Identification of the interaction between HTNV NP and DDX17The NP/DDX17 full-length and truncated plasmids containing the tag,namely Myc-NP/NP truncated plasmids,HA-DDX17/DDX17 truncated plasmids,were constructed according to the NP and DDX17 protein sequences and domains indicated by Uniprot.Then the following experiments were performed.（1）Myc-NP and HA-DDX17/DDX17 truncated plasmids were co-transferred into HEK-293T cells,and cell lysates were collected for co-immunoprecipitation（co-IP）after48 hpi（NP pull down DDX17/DDX17 truncated plasmids）.Immunoprecipitation（IP）were performed with anti-Myc-tagged antibodies,and immunoblotting（IB）with anti-HA-tagged antibodies,which was aimed at clarifying whether NP interacts with DDX17 and confirming the interaction domain of DDX17.The results indicate that NP can pull DDX17 down,and the aa314-652 region（DDX17-F3R4）of DDX17 is the key site for its interaction with NP.（2）Likewise,HA-DDX17 and Myc-NP/NP truncated plasmids were co-transformed into HEK-293T cells,and cell lysates were collected for co-IP experiments 48 h after transfection（DDX17 pull down NP/NP truncation）.IP is done with anti-HA tag antibody,and IB with anti-Myc tag antibody,which was aimed at clarifying whether DDX17interacts with NP and confirming the interaction domain of NP.The results suggest that DDX17 can pull NP down,and the aa121-429 region of NP is a key site for its interaction with DDX17.Previous literature reports that the aa314-652 region of DDX17 contains a helicase domain,suggesting that DDX17 may assist in the helicasing process of the HTNV genome;while the aa121-429 region of NP contains two RNA-binding domains,namely the RNA-binding domain aa121-300,which can specifically bind to viral RNA;non-specific RNA binding domain aa301-429,which can bind to any RNA and may play a role in the capture of host mRNA“hat”;and co-IP experiments show that DDX17 can interact which both of the two RNA binding domains individually,suggesting that DDX17 may play a role in viral genome helicasing and viral acquisition of host mRNA“hats”.2.4 Exploring the mechanism of sub-localization in cells of DDX17 during HTNV infectionThe Myc-NP and HA-DDX17 plasmids were transfected into HEK293T cells separately,and the cell were harvested as the sample 48 hpi.Then we used these samples for immunoprecipitation to detect the interaction between NP and KPNA or DDX17 and KPNA.The results show that both NP and DDX17 can bind KPNA1 and KPNA2,suggesting that NP may promote DDX17 molecules to remain in the cytoplasm by competitively binding KPNA1,2,which facilitates its viral replication.Conclusion（1）A number of host protein molecules interacting with NP,such as DDX17,were identified by affinity chromatography coupled with mass spectrometry.（2）DDX17 can promote the replication of HTNV,and the subcellular localization was found to accumulate in cytoplasm after HTNV infection.（3）The helicase domain of DDX17 interacts with the RNA binding domain and the unspecific RNA binding domain of NP,suggesting that DDX17 may assist in the helicasing process of the viral genome and acquiring the host mRNA“hat”to promote viral replication.（4）NP may cause DDX17 to accumulate in the cytoplasm by competitively binding KPNA1,2,thereby promoting HTNV replication.