Identification And Functional Analysis Of Host Factors FUBP3 And IFIT5 Associated With SFTSV Replication

Severe fever with thrombocytopenia syndrome(SFTS)was reported firstly in China in 2009.SFTS cases have been reported in more than 20 provinces in China,and in other Asian countries such as Japan and Korea.The causative pathogen is named Severe fever with thrombocytopenia syndrome virus(SFTSV).SFTSV infection causes fever,thrombocytopenia,gastrointestinal symptoms etc.with a high fatality rate of up to 30%,leading to a serious threat to public health.World Health Organization has listed SFTSV as one of the priority pathogens of research and development.Nucleocapsid protein(NP),a main structural protein of SFTSV,plays an important role in protecting viral genome,and mediating and participating in viral transcription and replication.However,the function and mechanism of how SFTSV NP utilizes host molecules to promote viral proliferation or conversely,how host molecules restrict virus replication are not yet clear.Therefore,we identified the host molecules potentially involved in SFTSV replication,and researched the functions and mechanisms of the identified host molecules,FUBP3 and IFIT5.This research expands the study of SFTSV-related host factors and identifies for the first time the regulatory role of FUBP3 on negative-strand RNA viruses.The transmission route,genomic constitution and replication strategy of SFTSV were described in detail in the first chapter.In addition,the previous research on SFTSV replication-related host molecules was briefly described,and the background of FUBP3 and IFIT5 was elaborated.Finally,the research content and significance of this thesis were summarized.Preliminary multi-omics analysis in our laboratory have uncovered a number of host factors that may regulate SFTSV replication,and Chapter 2 of this thesis focuses on further confirmatory analysis of potential SFTSV host interaction factors.Firstly,the eukaryotic expression vectors of the host factors constructed in the previous stage were confirmed,and additional eukaryotic expression vectors of the host factors were constructed to complete the preliminary identification at the level of the SFTSV minigenome system.The host factors that significantly affect the activation of the reporter system were further identified at the SFTSV infection level,and particularly two regulatory molecules of the viral replication,FUBP3 and IFIT5,were uncovered.Then the FUBP3 function was confirmed by overexpression,knockdown and knockout experiments at the cellular level.FUBP3 was found to have a dual regulatory effect on SFTSV replication: on the one hand,FUBP3 overexpression inhibited SFTSV replication;on the other hand,downregulation of FUBP3 expression also inhibited SFTSV replication.At the same time,SFTSV infection has a cell-specific regulatory effect on FUBP3 expression.Epithelial cells infected with SFTSV upregulated FUBP3 expression,while immune cells infected with SFTSV downregulated FUBP3 expression.Using interaction and immunofluorescence experiments at the infection and co-transfection levels,we found that FUBP3 not only interacted with the NP of SFTSV,but also with the NSs probably.Finally,we constructed truncated mutants of FUBP3,and identified KH3 as a key functional domain that is required for FUBP3 inhibition of the SFTSV minigenome system.We revealed for the first time the regulatory role of FUBP3 in negative-stranded RNA virus infections,deepening our understanding of the function of FUBP3.Meanwhile,the functional characteristics of FUBP3 suggest that it may be a promising therapeutic target for SFTS.In Chapter 3 of this thesis,we confirmed the antiviral function of IFIT5 using overexpression,knockdown and knockout experiments.IFIT5 overexpression inhibits SFTSV replication,and knockdown and knockout promotes SFTSV replication.IFIT5,a typical interferon stimulated gene,was found to induce upregulation of RNA and protein levels of IFIT5 by SFTSV infection,suggesting that the host may perform antiviral functions by increasing the expression of IFIT5.Protein interaction experiments by transfection revealed that IFIT5 interacts with NP and verified the protein interactions at the infection level.Co-localization of IFIT5 with the NP of SFTSV was also detected by immunofluorescence experiments.This study expands the function of IFIT5 and identifies and confirms for the first time the function of IFIT5 in the inhibition of SFTSV replication.In summary,we screened the host factors uncovered by the multi-omics analysis using SFTSV minigenome system,and the host factors that significantly influence the reporter system were further confirmed in the context of SFTSV infection,obtaining the key host factors FUBP3 and IFIT5.The molecular functions and targets of FUBP3 and IFIT5 in SFTSV infection were analyzed at the cellular level.These studies have extended the research of SFTSV-host interaction,and discovered for the first time the regulatory function of FUBP3 for negative-strand RNA viruses and IFIT5 inhibition for SFTSV,and deepened the understanding of SFTSV infection mechanism,expanding the molecular functions of FUBP3 and IFIT5.Meanwhile,the research in this thesis provides ideas for the study of other bunyaviruses and potential host targets for the design of antiviral drugs in the future.