Genome-Wide Gene Expression Analysis Identifies the Proto-oncogene Tyrosine-Protein Kinase Src as a Crucial Virulence Determinant of Infectious Laryngotracheitis Virus in Chicken Cells

Li, Hai; Wang, Fengjie; Han, Zongxi; Gao, Qi; Li, Huixin; Shao, Yuhao; Sun, Nana; Liu, Shengwang

Based on the aforementioned hypothesis, we predicted that ILTV infection would induce FAK activation in LMH cells. Immunoblotting results showed that the phosphorylation of Tyr 576/577 of FAK is enhanced by ILTV infection, although the constitutive phosphorylation of FAK at Tyr 397 and the total FAK levels were unaffected ( Fig. 6A to C). In line with the essential role of the phosphorylation of FAK at Tyr 397 in the Src-mediated activation of integrin-focal adhesion signaling (26) , the administration of a widely used FAK-specific chemical inhibitor, PF573228 (50) , not only downregulated the constitutive phosphorylation of FAK at Tyr 397 and prevented the phosphorylation of FAK at Tyr 576/577 but also blocked the phosphorylation of Src at Tyr 416 in ILTV-infected LMH cells (Fig. 6A) . These results support the involvement of FAK during ILTV infection and suggest that FAK activation is required for Src to regulate ILTV infection in LMH cells. Further immunoblotting results suggested that Src activity was indispensable for the phosphorylation of FAK at Tyr 576/577 during ILTV infection, because the phosphorylation of FAK at Tyr 576/577 during infection was prevented by pretreating the cells with either SU6656 (Fig. 6B) or specific siRNAs targeting Src (Fig. 6C) . The total FAK level was not significantly affected by Src inhibition. Taken together, the findings presented above suggest that there is a positive-feedback loop between Src and FAK during ILTV infection. Additionally, the constitutive phosphorylation of FAK at Tyr 397 in LMH cells was also down-regulated by SU6656 (Fig. 6B ) or specific siRNAs targeting Src (Fig. 6C) , indicating an essential role of basal Src activity in the maintenance of the constitutive phosphorylation of FAK at Tyr 397 in LMH cells.