Genome-wide Screening Of Key Host Factors For Porcine Transmissible Gastroenteritis Virus Replication And The Molecular Mechanism Research Of TMEM41B Regulates Coronavirus Replication

Coronaviruses（CoVs）have a wide range of hosts and many members.Severe Acute Respiratory Syndrome Coronavirus 2（SARS-CoV-2）recently caused a huge challenge to global public health and huge economic losses to the whole world.But the measures for preventing or treating CoVs infections were still limited.CoVs regulate the intracellular environment for efficient replication by hijack and employ the host protein.However,the related cellular host factors and the signaling pathways involved in the life cycle of CoVs are still not fully illuminated,and it is urgent for us to further explore the mechanisms.CRISPR library has proven to be a powerful tool for screening host factors required for infection by different viruses（SARS-COV-2,influenza A virus（IAV）,and Japanese encephalitis virus（JEV））.In this study,we used porcine CRISPR/Cas9 genome-wide knockout（KO）library to screen for host factors required for Transmissible gastroenteritis virus（TGEV）replication in PK-15 cells and focused on the mechanism of a key host factors TMEM41B for further study:1.A genome-scale CRISPR screen identified host factors associated with TGEV infection.We used alpha TGEV（MOI=0.001）as a model to perform three rounds genome-scale CRISPR KO screen.The 3 rounds of surviving mutant cells were collected for deep sequenced.ANPEP,the already known functional receptor of TGEV,was significantly enriched in all three rounds of CRISPR screening.These results suggested that the candidate host factors related to TGEV infection based on our CRISPR screening strategy are reliable.GO/KEGG functional annotation analysis was performed on the enriched target genes（reads>10,000 sg RNAs）,and it was found that the main biological processes involved included transforming growth factor receptor signaling,proteolysis,and glycosaminoglycan biosynthesis.To preliminarily verify the effect of candidate genes on TGEV replication,14 candidates（GRAMD1C,LPP,ARHGEF38,STK36,SEC24B,TRIM2,CMTM6,DYRK1A,AK8,PPP2R5D,TAX1BP1,VSIG2,BARHL2 and RPSA）were selected to construct knockout cell lines.It was found that different KO cell lines have different inhibitory effects for TGEV replication.2.Focused-CRISPR library were generated,screened and identified gene TMEM41B required for TGEV replication.To further explore the key host factors for TGEV replication,we generated a focused-CRISPR library which include the 79 top-ranked candidate host factors from the Pig Ge CKO screen（except for the sg RNA for ANPEP）,and we performed 5 rounds to screen with a higher virus titer（MOI=1）.4 sg RNAs were found to hit TMEM41B in the3 and 5 challenge rounds.Combined Genome-wide and Focused-CRISPR screen results,we constructed the five genes（ANPEP,TMEM41B,LPP,TAX1BP and BARHL2）KO cells.Virus titers and IFA results showed that the five KO cells all can inhibit TGEV replication.And the virus titers reduce 4 log on TMEM41B-KO cells.3.TMEM41B plays a vital role in the formation of CoVs replication organelle（RO）Transcriptome analysis and experimental results showed that TMEM41B as an immunonegative regulator,and this function is not involved in TGEV replication.The confocal results revealed that knockout TMEM41B inhibited the endocytosis of virions,and the extent of virion internalization decreased by approximately 30%.Moreover,the early replication stage of TGEV in TMEM41B-KO cell lines was also severely impaired.Ds RNA,a marker of TGEV replication,could not be formed in TMEM41B-KO cell lines.The TEM imaging was used to evaluate the ability of TGEV to form CoV RO in TMEM41B KO and WT cells.The results showed that typical double-membrane vesicles（DMVs）and other types of ROs including the small open double-membrane spherules（DMSs）and irregular coiled membrane formed by ERs expansion in WT cells upon infection with TGEV.However,we have not observed obvious DMVs and replication organelle structures modified by the virus in TMEM41B KO cells upon infection with TGEV.Meanwhile,we investigated the function of TMEM41B employed by the DMVs model that constructed by SARS-COV-2 non-structural protein（NSPs）,the results showed that the endoplasmic reticulum could not produce enough deformation to induce DMVs in HEK293T-TMEM41B-KO cell lines.These results suggested that TMEM41B plays an important role in the formation of CoVs replicating organelles.4.TMEM41B is a host factor required for the replication of multiple viruses from diverse familiesThe KO and rescue assays results showed that TMEM41B is an important host factor not only for CoVs（TGEV,Mouse hepatitis virus（MHV）and Porcine deltacoronavirus（PDCoV））replication,but also for flaviviruses（JEV）and negative RNA viruses（VSV and IAV）replication.5.TMEM41B is required for MHV infection in vivoTo evaluate TMEM41B as a host factor for CoV replication in vivo.TMEM41B^+/-mice were generated using CRISPR/Cas9 by the company.The KO mice infection model results showed that knockout of this factor can significantly inhibit viral infection and delay the progression of a CoVs disease.The viral titers in TMEM41B^+/-were significantly reduced compared with WT mice livers.These results support that TMEM41B is involved in CoV MHV infection in vivo.Thus,our studies screened and identified the key host factor TMEM41B used by genome-wide CRISPR/Cas9 technology,and systematically investigated the possible molecular mechanism of TMEM41B regulated CoVs replication.Our animal experiments demonstrated the functional relevance of TMEM41B for CoV infection in vivo for the first time,which indicates that TMEM41B might be a vulnerable target for the development of anti-CoV therapeutics.