The Molecular Mechanism Of Interferon-stimulated Genes TMEM41B And WARS2 In Promoting Pseudorabies Virus Proliferation

To ensure the optimal environment for replication and spread,viruses have evolved many strategies to hijack or reprogram host metabolic pathways,not only to meet their own material and energy needs for reproduction but also to evade host immune responses,ensuring virus replication.Therefore,a deep understanding of the molecular mechanisms behind this can provide important scientific theoretical basis for the prevention and control of viral infectious diseases.In previous studies,using Pseudorabies virus(PRV)to infectRAW264.7 cells,the research team analyzed the differentially expressed genes through transcriptomics and found that the m RNA expression of Transmembrane protein 41B(TMEM41B),involved in lipid synthesis,was upregulated.To explore the role of TMEM41 B in PRV proliferation,the upregulation of TMEM41 B m RNA and protein expression levels upon PRV infection was first validated.To reveal the molecular mechanism of PRV-induced upregulation of TMEM41 B expression,exogenous interferon-β(IFN-β)or natural immune activators(Poly(I:C),Poly(d A:d T),and HT-DNA)were added to the cell culture medium,which all upregulated TMEM41 B m RNA expression levels.Further studies found that PRV promotes TMEM41 B expression through the c GAS/STING/TBK1/IRF3 and type I interferon receptor(IFNAR1)pathways,indicating TMEM41 B as an interferon-stimulating gene(ISG).To clarify whether TMEM41 B is involved in PRV proliferation,TMEM41 B expression was knocked down using sh RNA,resulting in a significant decrease in virus proliferation,while overexpression of TMEM41 B promoted virus proliferation.To elucidate how TMEM41 B promotes PRV proliferation,the effect of TMEM41 B knockdown on the PRV life cycle was examined.The results showed that PRV entry into host cells was inhibited in TMEM41 B knockdown cells.Given the critical role of TMEM41 B in lipid synthesis,its role in PRV-induced lipid synthesis was analyzed.In control cells,PRV infection significantly promoted lipid content,while TMEM41 B knockdown inhibited PRV-induced lipid synthesis.However,supplementation of lipids in TMEM41 B knockdown cells restored the dynamics of membrane protein-coated vesicles required for PRV entry into host cells and virus proliferation,indicating that PRV promotes lipid synthesis by upregulating TMEM41 B,facilitating virus entry into host cells and proliferation.Further research revealed that TMEM41 B promotes lipid synthesis by upregulating the expression of key genes(SREBP1c,ACCA,FASN,SREBP2,HMGCR)and activating key enzymes(ACCA,FASN,HMGCR)involved in lipid synthesis.These results indicate that PRV infection upregulates TMEM41 B expression through the IFN signaling pathway,thereby promoting lipid synthesis and virus replication.Another gene upregulated in transcriptomics is Tryptophanyl-tRNA synthetase 2(WARS2),related to protein synthesis.Experimental results confirmed that PRV infection specifically upregulates WARS2 expression,and this process depends on c GAS/STING/TBK1/IRF3/IFNAR1.However,the expression of two other aminoacyl-tRNA synthetases,WARS1 and EARS2,is not regulated by PRV infection.IFN-β,Poly(I:C),Poly(d A:d T),and HT-DNA all activate WARS2 m RNA expression,indicating that WARS2 is also an ISG.Further research found that knocking down WARS2 inhibits PRV proliferation,while overexpression of WARS2 promotes PRV proliferation.Given the critical role of WARS2 in protein synthesis,its role in PRV-induced protein synthesis was analyzed.Knockdown of WARS2 inhibits PRV-induced protein synthesis,while overexpression of WARS2 promotes PRV-induced protein synthesis.Mutants of WARS2 with enzymatic activity(mut 48-54)and mutants lacking the mitochondrial targeting sequence(Δ1-18)cannot promote protein synthesis,indicating that PRV-induced protein synthesis depends on the enzymatic activity and mitochondrial localization of WARS2.Further research found that PRV infection promotes the formation of mitochondrial respiratory chain complexes,ATP,and acetyl coenzyme A through WARS2,and acetyl coenzyme A is a precursor molecule for lipid synthesis.The involvement of WARS2 in lipid synthesis was further investigated.The results showed that PRV infection through WARS2 also promotes the expression of key genes(SREBP1c,ACCA,FASN,SREBP2,HMGCR)and activates key enzymes(ACCA,FASN,HMGCR)involved in lipid synthesis.These results indicate that PRV infection upregulates WARS2 expression through the IFN signaling pathway,thereby promoting protein and lipid synthesis,facilitating virus replication.In summary,this study confirms that both TMEM41 B and WARS2 are ISGs and can promote PRV proliferation.This provides new targets and theoretical basis for the prevention and control of PRV.However,further in-depth research is needed to determine which of the two contributes more to PRV proliferation and whether they are functionally correlated.