Effects Of Infectious Laryngotracheitis Virus On Host Cells Metabolism And Mechanistic Study

Gallid alphaherpesvirus 1(GAHV-1),also known as avian infectious laryngotracheitis virus(ILTV),which belongs to the family Herpesviridae and the subfamily Alphaherpesvirinae and the only member of genus Iltovirus,is one of the main pathogens that are harmful to the poultry industry worldwide.Similar to human alphaherpesviruses,due to the establishment of latent infection in the trigeminal ganglion of the host after acute infection,ILTV cannot be cleared from the host and no effective therapeutic treatment is available currently.Virus-host metabolic interaction is a promising target for antiviral therapeutics.However,comprehensive exploration of virus-host metabolic interaction has only been limited to a few human herpesviruses and the metabolic requirements of ILTV remain largely unclear.To illustrate the basic metabolic requirements of ILTV,the present study used the ILTV-susceptible but nonpermissive chicken cell line DF-1 and the ILTV-permissive chicken cell line LMH as models,based on bioinformatic analysis of genome-wide transcriptome data by analysis of global metabolome assays of ILTV infected host cell lines in combination with a set of functional validations.We found that ILTV infection tended to promote a metabolic shift from glycolysis to fatty acid(FA)and nucleotide biosynthesis and utilizes glutamine independently of glutaminolysis,without significant general effect on the TCA cycle.In addition,the above metabolic requirements of ILTV infection revealed in chicken cell lines were further validated in chicken primary cells isolated from chicken embryo kidneys and chicken embryo livers.Moreover,different metabolic pathways were found to be required for distinct stages of ILTV replication.Glucose and glutamine were required for the transcription of viral immediate early gene ICP4 and subsequent steps of viral replication.However,FA synthesis was essential for assembly but not required for other upstream steps of ILTV replication.Like other herpesviruses,the expression of viral genes encoded by ILTV is under a cascade regulation pattern,whose underlying regulatory mechanism remains largely unclear.Viral immediate-early(IE)gene,ICP4,plays a prominent role in the initiation of the transcription of early and late genes during ILTV replication.In this study,we identified AP-1 as the key regulator of the transcription of ILTV genes by bioinformatic analysis of genome-wide transcriptome data.Subsequent functional studies of the key members of AP-1 family revealed that Fos,but not Jun,conferring the regulation of ILTV infection by AP-1,since knockdown of Fos,instead of Jun,by gene silencing significantly reduced ICP4 transcription and subsequent viral genome replication and virion production.Using several approaches,we identified ICP4 as a bona fide target gene of Fos with Fos response elements within its promoter,which confers the Fos regulation.While neither the physical binding nor the transcriptional activity of Jun to the promoter of ICP4 was observed.Besides,knockdown of Fos reduced the transcription of two host rate-limiting enzymes essential for the supplies of TCA intermediates OAA and ATP,MDH1 and ATP5A1.The biological significance of the transcriptional controls of MDH1 and ATP5A1 by Fos in ILTV infection was supported by the anaplerosis of OAA and ATP,which rescued both ICP4 transcription and virion production in infected cells under the condition of Fos-silencing.This study identified transcription factor Fos as a key regulator of ILTV infection through its transcription factor function on both virus and host sides,which might extend current AP-1for understanding of both avian herpesvirus and host interactions.Viral spread is considered a promising target for antiviral therapeutics.We previously identified proto-oncogene tyrosine-protein kinase Src(Src)as a crucial host determinant of ILTV infection.Our further study found that inhibition of Src to promote the intercellular transmission of ILTV was independent of viral replication,infected cell proliferation,extracellular vesicles,or direct cytoplasmic connections between cells.The effect of host fatty acid metabolism on human herpesvirus infection has been studied,and this study also confirms that fatty acid metabolism is necessary for ILTV assembly.To further clarify the spread mechanism of ILTV upon Src inhibition between the cells,the rate infection of ILTV-EGFP was detected by flow cytometry via tracing the EGFP signal without the supplementation fatty acid synthesis inhibitors TOFA or C75 in this study.We found that the presence of TOFA and C75 upon Src inhibition reduced the ILTV-infected positive cells and increased the death of infected cells,which could be reduced by the anaplerosis of PAL.Overall,these data suggest that Src controls the cell-to-cell spread of ILTV in a cellular fatty acid metabolism-dependent manner.Thus,this study demonstrate that fatty acid metabolism is essential for the regulation of the cell-to-cell spread and cytopathogenicity of ILTV by Src.Our previous study found that ILTV-infected cell inhibited p53 activity in uninfected cells through a paracrine-regulated pathway,while the activation of p53 not only prevented the apoptosis of remote uninfected cells and subsequent pathological damage induced by ILTV infection but also delayed viral dissemination significantly.As an important regulator of cell metabolism,the tumor suppressor p53,plays a key role in the cellular stress response.To analyze the function of p53 regulate ILTV-infected host cell metabolism,the present study used the ILTV-permissive chicken cell line LMH as models and explored the metabolic requirements of ILTV by preliminary analysis of global metabolome assays ILTV infected host cell.We found that inhibition of p53 increased the levels of metabolites related to cellular glycolysis,upstream of the TCA cycle,pentose phosphate pathway,and glutaminolysis.Activation of p53 can also increase the level of pentose phosphate pathway related metabolites,but the increase is less than that of p53 inhibition.These results suggest that p53 may regulate ILTV infection through cellular metabolic pathways of glycolysis,TCA cycle upstream,pentose-phophate pathway,and glutaminolysis.Our study provides a reference for extending our understanding of the roles of p53 in regulating cell metabolism to viral infection.In conclusion,this study uncovers the effect of ILTV infection on host cell metabolism and the regulation and mechanism of host regulatory factor Fos,Src,and p53 on cell metabolism during ILTV infection.Our study lays the foundation for a comprehensive analysis of the interaction between ILTV and host cells.