Study On The Mechanism Of The Regulation Of Infectious Laryngotracheitis Virus Infection By Host SRC

Avian infectious laryngotracheitis?AILT?is an upper respiratory disease of chickens caused by Infectious laryngotracheitis virus?ILTV?,which creates great economical losses worldwide annually.ILTV is a member of the Herpesviridae family and it can lead to latent infection,which protects it from host and results in the reactivation of ILTV from latency when host immunity is compromised.Therefore,novel strategies independent of host immunity are needed for AILT control.Targeting host-virus interactions is a promising strategy to combat ILTV.Our previous findings have showed that host SRC?proto-oncogene tyrosine-protein kinase?is a central modulator of ILTV infection,but it remains unclear that how it regulates ILTV infection.For such a purpose,time-lapse fluorescence microscopy via tracing ILTV expressing EGFP was performed with LMH cells upon SRC inhibition by two specific small molecule inhibitors,PP1 and PP2,and enhanced ILTV-EGFP transmission by SRC inhibition was observed.Furthermore,flow cytometry and Operetta CLS high-content analysis system results suggested that both the percentage and the area of EGFP-positive cells were increased significantly?p<0.05?after ILTV-EGFP infection upon SRC inhibition.Similar results were also gained by small RNA interference fragments specifically targeting different sequences of chicken SRC.As we all know that virus transmits by two ways,namely,cell-free transmission and cell-to-cell transmission.For better investigating the mechanisms of the enhanced ILTV transmission by SRC inhibition,the level of virus released into medium upon SRC inhibition was detected by qPCR assays and TCID₅₀ respectively.There was no significant difference between SRC inhibition and control and the level of ILTV in medium was even undetectable at 24 hpi.Importantly,Operetta analysis showed that the area of EGFP-positive cells was still increased significantly?p<0.05?with SRC inhibition at 24 hpi in the present of neutralizing antibody?against glycoprotein I of ILTV?.These findings suggested that SRC inhibition enhanced ILTV transmission independent on extracellular virions.Next,we examined viral binging,entry and transmission between adjacent cells.Flow cytometry result showed that SRC inhibition promoted penetration of ILTV but had no effect on binding.To further understand the mechanism by which SRC inhibition promotes cell-to-cell transmission,a co-culture system with infected donor cells and noninfected target cells was conducted.With this co-culture system,we found that the enhanced ILTV transmission by SRC inhibition was mainly triggered by SRC repression in donors independent of the proliferation of infected cells.Although naturally occurred between adjacent cells and was enhanced by ILTV infection,the direct cytosol-to-cytosol connectivity had no contribution to this process.To illustrate its mechanism on a molecular level,genome-wide transcriptional profile analysis was performed using cells with different SRC activities at 24 hpi.Subsequent bioinformatic analysis identified Akt,MEK and p38 MAPK pathways as the main molecular events required for the enhanced cell-to-cell transmission of ILTV by SRC inhibition.Further functional studies using small molecule inhibitors targeting abovementioned signaling pathways revealed that Akt and p38 MAPK pathways,but not MEK pathway,were required for the enhanced cell-to-cell transmission of ILTV by SRC inhibition.Our findings have provided us with new insights regarding the mechanisms by which host SRC regulates ILTV transmission,which advanced our understanding of host-ILTV interactions on a cellular and molecular level and also contributed to further mechanistic illustration of the cell-to-cell transmission induced by other alphaherpesviruses.