Studies On The Role Of Host Factor TMEM41B In PDCoV Proliferation

As an absolute intracellular parasitic microorganism,the effective proliferation of virus in host cells depends on the participation of a large number of host factors.PDCoV is a newly discovered porcine enterocoronavirus that mainly causes diarrhea and death of newborn piglets,causing certain economic losses to the pig industry.There is no safe and effective vaccine or specific therapeutic drug against this disease currently.Screening for host factors associated with PDCoV replication could provide a target for the development of anti-PDCoV drugs.On account of this,the whole pig genome was screened using the CRISPR-Cas9 knockout system in this study,and it was found that a multiple transmembrane protein TMEM41 B related to endoplasmic reticulum localization might be involved in the replication of PDCoV.The details are as follows.1.Screening of host factors related to PDCoV proliferationThe CRISPR-Cas9 sgRNA lentivirus library targeting the porcine whole genome was transduced into LLC-PK1 cells and then a heterozygous whole genome knockout cell library was obtained by pressure screening.The cell line was infected with PDCoV,after two rounds of screening,the genomic DNA extracted from the surviving cells was sequenced,and the sequencing results showed that TMEM41 B got a high score suggesting that TMEM41 B might be an important host factor in the proliferation of PDCoV.2.Knockdown of TMEM41 B inhibits PDCoV replicationIn order to verify whether TMEM41 B is involved in the replication of PDCoV,the si RNA targeting TMEM41 B was designed and synthesized and transfected into LLC-PK1 cells,and PDCoV was inoculated 24 h later.The RT-q PCR and Western-blot assay showed that knockdown of TMEM41 B inhibited the proliferation of PDCoV in LLC-PK1 cells,indicating that TMEM41 B was related to the proliferation of PDCoV in LLC-PK1 cells.3.Overexpression of TMEM41 B promotes PDCoV proliferationFurthermore,the eukaryotic expression plasmid of TMEM41 B was constructed and transfected into LLC-PK1 cells and then inoculated with PDCoV.RT-q PCR and Western-blot analysis showed that overexpression of TMEM41 B promoted the proliferation of PDCoV in LLC-PK1 cells,indicating that TMEM41 B did participate in the proliferation process of PDCoV in cells.4.Knockout of TMEM41 B significantly inhibits the proliferation of PDCoV,TGEV and PEDVDesign and synthesize two pairs of g RNAs targeting pig TMEM41 B exons No.1 and No.4,construct them into the PX-459 vector respectively,and transfect the constructed plasmid into LLC-PK1 cells.After pressure screening and sequencing,it was confirmed that two groups of TMEM41B-knockout cell lines KO-1(with one base inserted in TMEM41 B gene)and KO-2(with 10 bases missing in TMEM41 B gene)were obtained.The result of Western-blot confirmed that neither group of cells expressed TMEM41 B.The results of CCK8 kit showed that the knockout of TMEM41 B had no significant effect on cell activity.After inoculation with PDCoV,the indirect immunofluorescence,TCID50,and RT-q PCR assays showed that the knockout of TMEM41 B significantly inhibited the proliferation of PDCoV.After the overexpression of TMEM41 B on the knockout cell line,PDCoV proliferation was restored to some extent.The above results indicated that TMEM41 B was an important host factor related to the proliferation of PDCoV.On this basis,the TMEM41 B knockout cell line was used to detect the effect of knocking out TMEM41 B on the proliferation of TGEV and PEDV.The indirect immunofluorescence results showed that TMEM41 B knockout significantly inhibited the expression of TGEV S protein and PEDV S protein,indicating that knockout of TMEM41 B could also inhibit the proliferation of TGEV and PEDV.TGEV,PEDV and PDCoV all belong to the coronavirus family,and it is speculated that TMEM41 B may be an important host factor related to coronavirus infection.5.TMEM41 B is mainly involved in the replication step of PDCoV proliferationIn order to verify the specific mechanism of TMEM41 B participating in the proliferation of PDCoV,the TMEM41 B knockout cell line was used to detect the effects of knockout TMEM41 B on the attachment,internalization,replication and release step of PDCoV separately.The result of RT-q PCR showed that knockout TMEM41 B had no significant effect on the attachment step of PDCoV during its proliferation,but significantly inhibited the synthesis of PDCoV minus-strand RNA.The results of the plaque assay showed that knockout TMEM41 B had no significant effect on the internalization step of PDCoV proliferation,but it inhibited the release step of PDCoV to a certain extent.The above results indicated that TMEM41 B was mainly involved in the replication step and release step of PDCoV proliferation.6.TMEM41 B participates in PDCoV replication by influencing DMV formationThe effect of knockout of TMEM41 B on the formation of Double membrane vesicles(DMV)induced by PDCoV was observed under an electron microscope.The results showed that after knocking out TMEM41 B in LLC-PK1 cells,PDCoV could not induce DMV formation,but wild-type cells could induce the formation of DMV normally.In addition,Co-IP experiment confirmed the interaction between TMEM41 B and PDCoV nsp4,a scaffold protein of DMV.At the same time,we found that PDCoV infection would cause the intracellular distribution change of TMEM41 B from a diffuse to a punctate aggregation pattern.The above results indicated that TMEM41 B might participate in the replication phase of PDCoV by mediating the formation of PDCoV replication complex and DMV,and thus affect the proliferation of PDCoV.