Identification Of Host Factors Involved In SFTSV Infection Using CRISPR-based Screen

Severe fever with thrombocytopenia syndrome virus,SFTSV,a novel pathogenic bunyavirus,was first discovered in the Dabie mountain areas in China,clinically causing severe fever with thrombocytopenia syndrome with initial case fatality rate of 30%.Since the initial discovery of SFTSV,it has been reported in over 26 provinces in China and this expanding endemic of SFTSV infection has become a more important global public concern.Up to date,there have been currently no vaccines or specific antivirals available against SFTSV infection.As a high-efficient,stable and simple tool,CRISPR/Cas9 system allows easy knockout both alleles in a substantial percentage of treated cells and it has recently been developed as a tool for gene editing in mammalian cells.Using a high-complexity,genome-wide sgRNA library,the CRISPR-based screen has wide potential use to screen host cell factors which may play important role in various intracellular trafficking pathways during the process of viral infection.For HCV,Dengue virus(DENV),Japanese encephalitis(JEV),and West Nile virus(WNV),a number of genes associated with endoplasmic reticulum(ER)pathways and virus receptor have been screened out.Screens for other viruses,such as Human immunodeficiency virus(HIV)and influenza viruses,have identified host factors involved in virus entry,sialic acid biosynthesis and related glycosylation pathways.However,nearly no screens on bunyavirus infection were performed,and many host proteins involved in various intracellular pathways for their infection still remain unclear.Using CRISPR-based arrayed screen performed in 96-well plates,we identified SNX11 as an essential host factor for SFTSV infection.Then,a SNX11 knockout HeLa cell line was generated using CRISPR/Cas9 technology,and the decreased infection rate was validated in SNX11-KO cells and was rescued by re-expression of SNX11.To explore the function of SNX11 in SFTSV infection,we performed a confocal microscopy analysis which showed that the intracellular trafficking of SFTSV GP was blocked inside the endolysosomes,and SFTSV GP could not detected in ER and Golgi apparatus.These results indicated that the penetration of SFTSV into the cytoplasm was blocked and thus led to a decrease of SFTSV infection rate.It was well known that the essence of viral penetration is the fusion between the viral envelope and the endosomal membrane and for most of bunyaviruses,acidification has been shown to be sufficient to trigger the membrane fusion.Using flow cytometry analysis,we found that the pH inside the endosomal compartments of SNX11-KO cells increased from 5.33 to 5.56,and we speculated that this slight change was sensitive enough to affect the membrane fusion,thus to block the penetration of SFTSV.Besides,our results suggested that the lysosomal-associated membrane protein 1(LAMP1)expression was significantly elevated in the SNX11-KO cells.Then,we performed an electronic microscopy analysis and the results showed that the endolysosomes increased in SNX11-KO cells.Finally,using a RNA sequencing,we found that the knockout of SNX11 led to expression changes of many proteins that mainly located in Endocytosis pathways,which can provided important clues for the position where SNX11 works.In brief,we for the first time used CRISPR-based screen to study host factors for SFTSV infection,and we identified SNX11 as an essential gene for SFTSV infection.Moreover,we investigated the function of SNX11 in SFTSV infection process.However,the molecular functions of SNX11 in intracellular trafficking pathway are poorly characterized,and more solid evidence are needed to investigate the interactions,regulations of this cellular protein in SFTSV entry process.We also believe that SNX11 may play an important role in the infection processes of other enveloped viruses,which requires urgent attention in further research.