DEAD-box RNA Helicase 1(DDX1)as A Host Restriction Factor Against SFTSV And SARS-CoV-2 Replication And Screening Of Antiviral Drug

Historically,RNA viruses have caused numerous human and animal public health events,such as 1918 flu pandemic,2003 SARS,2014 Ebola epidemic in West Africa,and the recent COVID-19,which have seriously threatened human health and hindered socioeconomic development.Due to the high genetic variability of RNA virus,it is difficult to develop vaccines and drugs.In this study,we focused on two typical emerging RNA viruses,severe fever with thrombocytopenia syndrome virus(SFTSV)and severe acute respiratory syndrome coronavirus 2(SARS-Co V-2),in order to investigate the mechanisms of virus-host interactions.SFTSV,a novel tick-borne bunyavirus first discovered in China in 2009 with wide distribution,high pathogenicity and increasing incidence year by year.And the on-going pandemic of coronavirus disease 2019(COVID-19)caused by SARS-Co V-2has led to unprecedented medical and socioeconomic crises.Although there have been many studies on the molecular mechanisms of these two viral infections,the details of SFTSV and SARS-Co V-2 infection and pathogenesis are still largely unclear and need to be further investigated.The study of virus-host interactions may improve the understanding of viral pathogenesis and may provide clues for the screening of drug candidates.Our study focuses on the interaction of the host factor DEAD-box RNA helicase 1(DDX1)with the above two emerging RNA viruses,especially the viral nucleocapsid(N)protein.N,a key structural protein of virus,is involved in viral transcription and replication,and may regulate or are regulated by host proteins and the corresponding biological processes,leading to wide effects on viral infection and pathogenesis.Accordingly,the N protein has been also exploited as one of the therapeutic targets for antiviral research.DDX1 is an ATP-dependent RNA helicase and participates in RNA metabolism.It has been reported to play negative or positive regulatory roles in some virus infections.Currently,the interactions of DDX1 with SFTSV and SARS-Co V-2 are still unclear.Considering the critical roles of N,we focus on the N-cell interactions.The details are as follows:Here,we focused on the interaction between SFTSV and the host factor DDX1.The gain/loss-of-function experiments showed that DDX1 likely acts as potent anti-SFTSV host factors,inhibiting the viral replication.Mechanistic studies revealed that the C-terminal domain of DDX1 may be the key domain for anti-SFTSV activity.Furthermore,the anti-SFTSV activity of DDX1 might be independent on its ATPase and RNA helicase activities.In DDX1 knockout cells,SFTSV-induced IFN-β and TNF-α levels were low,suggesting that DDX1 might inhibit SFTSV by inducing interferon and inflammatory responses.The interaction between SFTSV and DDX1 showed that DDX1 interacts with N in an RNA-independent way and that the D1 domain of DDX1 might required for interaction with N.SFTSV minigenome reporter system suggested that DDX1 might affect RNP activity.This indicated that DDX1 might directly target N and affect its function,thereby inhibiting SFTSV.NSs of SFTSV,as an important virulence factor,could hijack DDX1 into the inclusion body(IBs)and interfere with its antiviral function,and the detailed potential function and mechanism need further investigation.In conclusion,DDX1 inhibition of SFTSV infection might has two main strategies:(1)DDX1 might mediate antiviral interferon and inflammatory responses.(2)DDX1 might directly target SFTSV N and affect its function.Meanwhile,SFTSV may use NSs to hijack DDX1 into the "inclusion body jail" to escape the antiviral function of DDX1.In the next chapter of this thesis,we established a new cellular interactome of SARS-Co V-2 N by using a high-specific affinity purification(S-pulldown)assay combined with liquid chromatography-mass spectrometry(LC-MS).Bioinformatics analysis showed that these host factors are mainly involved in translation regulations,viral transcription,RNA processes,stress responses,protein folding and modification,and inflammatory/immune signaling pathways,which are consistent with the putative role of N in viral infection.Through bioinformatics analysis and literature search,the possible pharmacological cellular targets and the directing drugs were mined,generating a drug-host protein network.A new antiviral small molecule drug,anisomycin,was screened and identified accordingly,and it was found to effectively inhibit SARS-Co V-2 replication,showing a dose-dependent effect(IC50=6.785 μM)and low cytotoxicity(CC50>1000 μM).In addition,DDX1 was verified to interact with N probably by binding to multiple domains of the viral protein.Loss-of-function experiments then showed that DDX1 likely acts as potent anti-SARS-Co V-2 host factors,inhibiting the viral replication and protein expression.These data provide new clues to further depiction of N-cell interactions and SARS-Co V-2 infection and may help develop new therapeutic candidates.In conclusion,this study focused on the viral N-cell interactions,and revealed the interaction mechanism between host factors DDX1 and N,which has broadened the understanding of the function of host factor DDX1 and provided insights into the interactions of SFTSV and SARS-Co V-2 with host cells.And it will facilitate not only the elucidation of infection and pathogenesis but also the development of antiviral intervention therapies.