key: cord-0002077-qwrdr92h
authors: Li, Hai; Wang, Fengjie; Han, Zongxi; Gao, Qi; Li, Huixin; Shao, Yuhao; Sun, Nana; Liu, Shengwang
title: 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
date: 2015-12-17
journal: J Virol
DOI: 10.1128/jvi.01817-15
sha: 6c3132687a4cd25b59f39f851b6b924c99afe345
doc_id: 2077
cord_uid: qwrdr92h

Given the side effects of vaccination against infectious laryngotracheitis (ILT), novel strategies for ILT control and therapy are urgently needed. The modulation of host-virus interactions is a promising strategy to combat the virus; however, the interactions between the host and avian ILT herpesvirus (ILTV) are unclear. Using genome-wide transcriptome studies in combination with a bioinformatic analysis, we identified proto-oncogene tyrosine-protein kinase Src (Src) to be an important modulator of ILTV infection. Src controls the virulence of ILTV and is phosphorylated upon ILTV infection. Functional studies revealed that Src prolongs the survival of host cells by increasing the threshold of virus-induced cell death. Therefore, Src is essential for viral replication in vitro and in ovo but is not required for ILTV-induced cell death. Furthermore, our results identify a positive-feedback loop between Src and the tyrosine kinase focal adhesion kinase (FAK), which is necessary for the phosphorylation of either Src or FAK and is required for Src to modulate ILTV infection. To the best of our knowledge, we are the first to identify a key host regulator controlling host-ILTV interactions. We believe that our findings have revealed a new potential therapeutic target for ILT control and therapy. IMPORTANCE Despite the extensive administration of live attenuated vaccines starting from the mid-20th century and the administration of recombinant vaccines in recent years, infectious laryngotracheitis (ILT) outbreaks due to avian ILT herpesvirus (ILTV) occur worldwide annually. Presently, there are no drugs or control strategies that effectively treat ILT. Targeting of host-virus interactions is considered to be a promising strategy for controlling ILTV infections. However, little is known about the mechanisms governing host-ILTV interactions. The results from our study advance our understanding of host-ILTV interactions on a molecular level and provide experimental evidence that it is possible to control ILT via the manipulation of host-virus interactions.

I nfectious laryngotracheitis (ILT) is a major respiratory disease of chickens that is induced by avian ILT herpesvirus (ILTV; also known as gallid herpesvirus 1). ILTV belongs to the Iltovirus genus within the Herpesviridae family. Infection with ILTV can induce a mild to severe upper respiratory tract disease in chickens, depending on the virulence of the virus, and it causes large economic losses to the poultry industry worldwide annually. Vaccination using live attenuated virus has been the most commonly used method to control ILT outbreaks since the mid-20th century. Vaccination infects naive chickens with live attenuated virus, thereby establishing latent viral carriers; however, it is not recommended that this vaccine be used in regions where ILT outbreaks are absent (1) (2) (3) (4) . The majority of currently used live attenuated virus vaccines can induce successful protection in chickens (5) . New outbreaks of ILT are continually being identified in many countries, especially in regions where live attenuated vaccines have been extensively administered (6) . A growing body of evidence from molecular typing data collected worldwide has revealed that the currently used live attenuated vaccine strains could generate, via recombination, a virulent virus that could be the cause of current outbreaks (7) (8) (9) (10) (11) (12) . Increasing concerns regarding the biosafety of live attenuated ILTV vaccines have triggered the development of recombinant vaccines using viral vectors, and these have been released in some countries (6) . However, none of the currently available recombinant vaccines are able to clear latent virus from an infected host. As a consequence, a residual latent infection can induce new outbreaks under certain conditions when host immunity is compromised, such as under stress and at the onset of egg laying (13, 14) . Therefore, novel strategies that are independent of the host immune system, such as tighter farm biosecurity and anti-ILTV breeding, are required to control ILT outbreaks. It is hoped that these new strategies that are being developed will improve the control of ILT and reduce the extensive use of live attenuated virus as vaccines.

In addition to controlling ILTV infections by establishing cellextrinsic barriers that are mediated by anti-ILTV innate and cellmediated immune responses (15) (16) (17) , the manipulation of the cell-intrinsic barrier that is mediated by host factors that regulate the interaction between ILTV and host cells could be another promising option. Many studies have investigated herpesvirushost interactions in humans and mice; however, little is known about these interactions in chickens. Lee et al. explored the molecular events induced by ILTV infection in host cells using publicly available high-throughput data on host transcription profiles in chicken embryo lung cells infected with a virulent ILTV strain (18) . A bioinformatic analysis revealed that several genes and pathways are significantly altered by ILTV infection; these findings were validated using quantitative PCR (qPCR) assays. Their study cast the first light on the molecular nature of the host response to ILTV infection. However, the underlying molecular basis of the host response remains largely unclear, and additional studies are urgently needed to address this issue.

The proto-oncogene tyrosine-protein kinase Src (Src) was originally isolated from a chicken sarcoma, and it was the first oncogene to be identified (19) . It is well-known that Src is an important factor for a wide range of biological processes, such as cell proliferation, adhesion, angiogenesis, organization of the cell skeleton, cell division, and cell death (20) (21) (22) (23) (24) . The tyrosine kinase focal adhesion kinase (FAK) has been shown to be required for integrin-stimulated cell migration and focal adhesion formation (25) (26) (27) , the disruption of which triggers apoptosis (28, 29) . The interaction between Src and FAK has been shown to promote the entry of human herpes simplex virus 1 (HSV-1) into target cells (30, 31) . However, to the best of our knowledge, the effects of host Src on host cell death during human or animal herpesvirus infections have never been reported, despite the fact that Src is considered to be both a survival factor (32, 33) and a death factor (33) (34) (35) .

In the present study, we used Leghorn male hepatoma (LMH) cells infected with ILTV as an in vitro experimental model. We investigated the molecular mechanisms of host cell responses to ILTV infection using genome-wide transcriptome studies in combination with bioinformatic analyses. We also conducted functional studies to examine the interaction between host Src and FAK.

Ethics statement. Animal experiments were approved by the Animal Ethics Committee [approval no. SYXK (Hei) 2011022] of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (CAAS), and they were performed in accordance with ethics guidelines using approved protocols.

Experimental animals. The allantoic cavity of 9-day-old specificpathogen-free (SPF) chicken embryos was inoculated with 100 l of virus specimens at 1 ϫ 10 4 or 1 ϫ 10 5 50% tissue culture infective doses (TCID 50 ) per ml, as indicated below, as well as 25 to 250 ng of SU6656 (Selleckchem, Houston, TX, USA), 250 ng of PF573228 (Sigma-Aldrich, St. Louis, MO, USA), or an equal volume of dimethyl sulfoxide (DMSO). All chicken embryos were incubated at 37°C and examined daily over 5 days. The chorioallantoic membranes (CAMs), allantoic fluids, and livers were then harvested, homogenized, and subjected to three cycles of freezethawing. Hematoxylin and eosin (H&E) staining was performed on sections of liver samples fixed with 4% paraformaldehyde as previously described (36) .

Virus strain and cell line. The LJS09 strain of ILTV (GenBank accession no. JX458822) (15) is a virulent strain of ILTV that was isolated in China, and its genome has been sequenced. It was isolated in 2009 from 6-week-old Yellow-Foot layers housed on a farm in Jiangsu Province, China, which had not been vaccinated against ILTV, and it was stored at the Harbin Veterinary Research Institute of the CAAS. This strain can be propagated in a chemically immortalized LMH cell line, in which clear cytopathic effects (CPEs) have been observed (37, 38) . LMH cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. Cell cultures were incubated at 37°C in 5% CO 2 . The Src inhibitor SU6656 and the FAK inhibitor PF573228 were used at 1 and 10 M, respectively, for in vitro experiments. Control groups were treated with DMSO at the same concentrations.

RNA interference and transfection. Two short interfering RNAs (siRNAs) that specifically recognize different sequences of the SRC mRNA (GenBank accession no. NM_205457.2; siSRC #1, 5=-CCA AAC ACG CUG AUG GCU U-3=; siSRC #2, 5=-GCC UCC UGG AUU UCC UGA A-3=) and a control siRNA (siControl; 5=-GCA CUU GAU ACA CGU GUA A-3=), which does not have a specific target site in chickens, were used. Transfection of siRNAs was conducted using an N-TER nanoparticle siRNA transfection system (Sigma-Aldrich) according to the manufacturer's instructions.

Plaque assay. Liquid medium-based plaque assays were performed as previously described (39) , with minor modifications. Briefly, LMH cells were exposed to virus that was diluted in serum-free DMEM for 3 h at 37°C in 5% CO 2 and then washed with phosphate-buffered saline (PBS). The cells were subsequently covered with DMEM containing 10% FBS, fixed with 70% ethanol at each time point postinfection indicated below, and stained with 0.1% crystal violet.

Quantitation of virus. LMH cells were infected with LJS09 at a multiplicity of infection (MOI) of 0.1. The indicated MOI was obtained according to the number of cells to be infected and the number of infectious particles, which were estimated on the basis of the numbers of PFU, which were calculated using the following formula: TCID 50 ϫ 0.59 Ϸ PFU [1 TCID 50 is equal to Ϫln(0.5) PFU and is approximately 0.59 PFU] (40). Levels of virus replication were determined using TCID 50 assays (40) on LMH cells and ILTV-specific qPCR assays (38) as previously described.

Flow cytometry and immunofluorescence. We conducted fluorescence-activated cell sorting (FACS) analyses using a BD FACScan flow cytometer and CellQuest software (version 4.0.2; BD, Mountain View, CA, USA). Cell death was assayed by examining cells in the sub-G 1 phase of the cell cycle via propidium iodide (PI) staining of permeabilized cells as previously described (41) . Apoptosis and necrosis were assayed with annexin V-PI using an annexin V-PI staining kit (Biyuntian, China) according to the manufacturer's instructions. Immunofluorescent detection of ILTV-positive cells was conducted on cells that had been fixed with 4% paraformaldehyde overnight. Fixed cells were incubated with a rabbit polyclonal antibody against ILTV glycoprotein I (gI), followed by a secondary goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC) (The Jackson Laboratory, Bar Harbor, ME, USA). A polyclonal antibody against the gI of ILTV was produced in a rabbit. Briefly, the rabbit was first immunized with the pCAGGS-gI vector via intramuscular injection, followed by boosting with inactivated ILTV and recombinant gI protein via intramuscular injection. The background level of reactivity was determined using control serum from nonimmunized SPF rabbits.

Protein extraction and Western blotting. Western blotting was performed under reduced denaturing conditions according to previously described procedures (42) . Antibodies against Src (EMD Millipore, Billerica, MA, USA), phosphorylated Src Tyr416 (EMD Millipore), FAK (Santa Cruz, Shanghai, China), phosphorylated FAK Tyr397 (Cell Signaling Technology, Inc.

[CST], Shanghai, China), phosphorylated FAK Tyr576/577 (CST), ␤-actin (Sigma-Aldrich), and ␤-tubulin (Sigma-Aldrich) were used. The intensity of the bands was measured using ImageJ software (43) .

To profile mRNA expression in LMH cells, we used a 4 ϫ 44K chicken gene expression microarray (catalog number 026441; Agilent Technologies, Santa Clara, CA, USA) as previously described (44) . We conducted qPCR assays as described previously, using specific oligonucleotide primers (Table 1 ) (44) . Data were analyzed with the 2 Ϫ⌬⌬CT threshold cycle (C T ) method, and the results are presented as the log 2 fold change.

High-throughput data analysis. Microarray data were analyzed with R (http://www.r-project.org/). Gene ontology (GO) analysis and pathway analysis were performed with DAVID bioinformatics resources (gene enrichment analysis using the EASE score with a modified Fisher's exact test P value of Ͻ0.05 as the threshold) (45) . Functional protein network analysis was performed with STRING software (46) .

Histopathological examination. Samples of embryo livers were collected and fixed in 4% paraformaldehyde. The fixed samples were sent to the Plague Diagnosis and Technical Service Center of the Harbin Veterinary Research Institute (an animal biosafety level 3 facility accredited by the China National Accreditation Service for Conformity Assessment [CNAS]) for histopathological examination. Briefly, samples were em-bedded in paraffin and then sectioned. The sample slides were stained with H&E and observed by light microscopy. A histopathological examination report was provided by the Plague Diagnosis and Technical Service Center.

Statistical analysis. We used the SPSS software package (SPSS for Windows, version 13.0; SPSS Inc., Chicago, IL, USA) for statistical analysis. Data obtained from several experiments are reported as the mean Ϯ standard deviation (SD). The difference between two groups was determined by Student's t test. One-way analysis of variance (ANOVA) with a Bonferroni correction was employed for multigroup comparisons. For all analyses, a P value of Ͻ0.05 was considered significant.

Microarray data accession number. Microarray raw data were uploaded to the National Center for Biotechnology Information Gene Expression Omnibus database under accession number GSE70855.

The biological characteristics of ILTV-LSJ09 infection in LMH cells, including cell growth, cell death, virus replication, virus release, and virulence, were examined. ILTV infection significantly reduced the number of adherent cells that were negative for trypan blue staining by the third day postinfection (3 dpi), while cells in the control cultures continued to grow (Fig. 1A) . Consistent with the reduction in the proportion of living cells following ILTV infection in our system (Fig. 1A) , the proportion of apoptotic cells significantly increased from 2 dpi compared with that of control cells (Fig. 1B) . The CPE of ILTV on LMH cells occurred by 2 dpi and became more severe as time progressed (Fig.  1C ). In parallel with the induction of apoptosis and the occurrence of the CPE, the results of the qPCR assay showed that ILTV replicated exponentially in LMH cells from 2 dpi, whereas viral replication was not significant at 1 dpi compared with that at 0 dpi (Fig .  1C ; the P values for replication at 1 and 2 dpi were 0.127 and 0.022, respectively). In addition, a concomitant, continuous increase in the copy number of the viral genome was observed in the medium from 2 dpi (Fig. 1C) , which possibly explains the occurrence of the ILTV CPE at 2 dpi (Fig. 1C) . The results of the qPCR assays were confirmed by TCID 50 assays (Fig. 1D ). No significant CPE was observed until 2 dpi, after which the virus titer increased continually in the cells and medium (Fig. 1D ). The proportion of ILTVinfected cells was determined by FACS, and it was approximately 10% at 2 dpi but only about 1.5% at 1 dpi (Fig. 1E ).

To understand the molecular events induced by ILTV infection in LMH cells when the CPE was evident, RNA was isolated from LMH cells at 2 dpi instead of at 1 dpi. We chose this time because the proportion of virus-infected cells at 1 dpi was too low to differentiate the transcription signature of virus-infected cells from that of all cells ( Fig. 1C and D). We identified 608 genes that were differentially expressed between control and ILTV-infected cells based on the following criteria: (i) a P value of Ͻ0.001 and (ii) a fold change in expression of Ͼ1.5 (data not shown). Hierarchical clustering analysis using these 608 genes demonstrated efficient clustering of biological replicates for control and ILTV-infected cells. We identified 297 downregulated genes and 311 upregulated genes following infection ( Fig. 2A) . For validation, the transcription levels of 20 genes which were randomly selected from the 608 genes were examined using qPCR assays. The qPCR results corresponded with those from the microarray assays (Fig. 2B) .

GO analysis was performed using the DAVID functional an- 

notation (subset GOTERM_CC_FAT, GOTERM_BP_FAT, and GOTERM_MF_FAT) (45) with a P value of Ͻ0.05. The five functions from each subset showing the greatest differential gene expression are listed in Fig. 2C . Cellular component analysis revealed that these differentially expressed genes were significantly enriched in those involved in the extracellular region and the plasma membrane (Fig. 2C) . Meanwhile, after ILTV infection the major biological processes controlled by genes that were differentially expressed were the regulation of cell proliferation, the enzyme-linked receptor protein signaling pathway, skeletal system development, and embryonic organ development (Fig. 2C) . The molecular functions of these differentially expressed genes appeared to be focused toward serine peptidase activity (Fig. 2C ).

Our GO analysis indicates that serine peptidase-based regulation of cell proliferation, the cytoskeleton system, and development through extra-and intracellular signal transduction may be the primary molecular events that occur in LMH cells upon ILTV infection.

To determine the central modulator of the molecular network in LMH cells after ILTV infection, we analyzed the functional connections between the proteins encoded by the 608 significant genes using STRING software with the default settings. The protein-protein interaction network was visualized by use of the Cytoscape program (47) , with the layout of group attributes being done according to the degree of connectivity of the nodes (Fig.  2D ). Each green rectangle represents a protein whose mRNA expression was altered by ILTV infection, and these are arranged in order of their functional connectivity to the other proteins in Fig.  2D , with the most highly connected proteins being on the top line and the least connected ones being at the bottom of Fig. 2D . Most of these nodes were directly or indirectly connected to Src, plasminogen (PLG), and serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1), indicating that these three factors were the central regulators. PLG and SERPINC1 were downregulated by ILTV infection, while Src was upregulated ( Src is a key determinant of ILTV virulence and replication in LMH cells. In ILTV-infected LMH cells, Src phosphorylation was greatly increased, although there was only a minor change in the Src protein level (Fig. 3A) .

Virus-induced cell death results in extensive tissue damage during the late stages of a virulent virus infection. We sought to The percentages of ILTV-infected LMH cells at 1 dpi and 2 dpi were measured by flow cytometry using a polyclonal antibody against glycoprotein I of ILTV, which was produced in SPF rabbits, followed by a fluorescein isothiocyanate-conjugated anti-rabbit second antibody. The level of background staining was determined using normal SPF rabbit serum as a control. Data are presented as the mean Ϯ SD (n ϭ 3). *, P Ͻ 0.05; N.S., no significant difference. determine whether Src had any effect on ILTV-induced cell death using two strategies. First, the depletion of Src halted ILTV-induced Src phosphorylation at Tyr 416 (Fig. 3A) , resulting in the promotion of the CPE (Fig. 3A) . This effect was also evident in LMH cells that were pretreated with a well-characterized Src-spe-cific chemical inhibitor, SU6656 (48) (Fig. 3B) , which interrupts Src phosphorylation upon ILTV infection (Fig. 3B) . FACS analysis of PI-stained cells found that ILTV infection in combination with the Src inhibitor resulted in levels of cell death at 2 dpi that were 4-fold higher than those in cells infected with ILTV alone (Fig. 3C) . A further detailed analysis using annexin V-PI staining revealed a significant enhancement of necrosis and apoptosis in ILTV-infected cells upon either siRNA-mediated Src depletion or the administration of SU6656 (Fig. 3D and E) , thereby demonstrating that inhibition of Src promoted LMH cell death during ILTV infection. The reduced threshold of ILTV-induced cell death by Src inhibition suggests that Src prolongs the survival of LMH cells in response to ILTV infection. Along with greater ILTV virulence, inhibition of host Src by either siRNA-mediated Src depletion or the administration of SU6656 resulted in Ͼ60% reductions in virus replication ( Fig. 3F and G, right) , as determined by qPCR when all virus-infected cells were dead ( Fig. 3F and G, left) . were validated in ovo using 9-day-old SPF chicken embryos that were inoculated through the allantoic cavity with 1 ϫ 10 4 TCID 50 of ILTV. Under our experimental conditions, inoculated embryo deaths occurred by 9 dpi, and all inoculated embryos were dead by 12 dpi (see Fig. S2 in the supplemental material). We first inoculated embryos with ILTV in combination with a serial dilution of the Src inhibitor SU6656. For the entire observation period (5 dpi), dead embryos were not observed at the lowest dose of SU6656 tested (25 g). A dose of 50 g of SU6656 resulted in the deaths of inoculated embryos by 2 dpi, with the mortality rate approaching 50% at 5 dpi (Fig. 4A) . The highest dose of SU6656 (250 g) resulted in the deaths of inoculated embryos by 1 dpi; all inoculated embryos were dead by 3 dpi (Fig. 4A) . During the observation period, there were no embryo deaths in eggs treated with ILTV alone or in uninoculated eggs treated with the highest dose of SU6656 alone (Fig. 4A) . Thus, the in ovo CPE that we observed was due to the combined effects of ILTV infection and SU6656 administration. Virus replication in allantoic fluids or CAMs was quantified by qPCR assays. Virus replication was reduced by SU6656 in a dose-dependent manner in allantoic fluids and CAMs ( Fig. 4B and C) .

Next, to determine whether the effects of the Src oncogene on ILTV infection that we observed in the chicken hepatoma cell line LMH could also be observed in normal liver cells, an in ovo examination of embryo livers was conducted. Histopathological examinations of slides with liver tissue stained with H&E revealed the infiltration of lymphocytes in all inoculated embryos (data not shown). However, different levels of hepatic tissue damage were observed among the inoculated embryos upon the administration of serially increasing dosages of SU6656. In embryos inoculated only with ILTV, necrosis was absent in four of the six samples, and mild necrosis was observed in the other two (Fig. 4D) , while hepatic tissue necrosis occurred in most of the inoculated embryos upon coinoculation with SU6656 (with 25 g of SU6656, tissue necrosis was absent in three embryos, mild in one embryo, moderate in one embryo, and severe in one embryo; with 50 g of SU6656, tissue necrosis was absent in two embryos, mild in one embryo, moderate in one embryo, and severe in two embryos; with 250 g of SU6656, tissue necrosis was moderate two embryos and severe in four embryos) (Fig. 4D) . No necrosis was observed in the livers of either control embryos or embryos that were inoculated only with 250 g of SU6656 (Fig. 4D) . Consistent with the findings in LMH cells (Fig. 3F and G) , embryo allantoic fluids (Fig.  4B) , and CAMs (Fig. 4C) , a dose-dependent reduction of virus replication by SU6656 was also observed in inoculated embryos, as assayed by qPCR (Fig. 4E) .

To explore the effect of virus dosage on the virulence and replication of ILTV in ovo, 9-day-old SPF chicken embryos were inoculated with high (1 ϫ 10 4 TCID 50 ) or low (1 ϫ 10 3 TCID 50 ) doses of ILTV with or without the administration of 100 g of SU6656. All embryos inoculated with the high viral dose in the presence of 100 g of SU6656 were dead by the end of the observation period, while no embryo deaths occurred in any other group (Fig. 5A) . SU6656-mediated reductions of viral replication were observed in the allantoic fluids (Fig. 5B) , CAMs (Fig. 5C) , and livers (Fig. 5D ) of embryos inoculated with either dose of ILTV, which rules out the possibility that SU6656 did not affect viral replication in embryos inoculated with a low dose of virus, thereby demonstrating that the difference in survival rates between the embryos inoculated with high or low doses of ILTV (in combination with 100 g of SU6656) resulted from the different sizes of the viral inoculum.

A pathway analysis of the subgroup of differentially expressed genes most closely connected to Src (see Fig. S3A in the supplemental material) indicated that the integrin signaling, focal adhesion, and adherens junction pathways were directly regulated by Src (see Fig. S3B in the supplemental material). Despite being involved in a wide range of biological processes (20) (21) (22) (23) (24) , it is well-known that Src mediates integrin-focal ad- hesion signal transduction by interacting with FAK (27, 49) . Thus, we hypothesized that activated FAK is required for Src activation and the subsequent prolonged survival of LMH cells following ILTV infection.

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.

Blocking FAK activity through the use of PF573228 exacerbated the ILTV CPE (Fig. 7A) . The level of apoptosis induced by ILTV in cells pretreated with PF5732228 was nearly 2-fold higher than that in DMSO-treated cells at 2 dpi (Fig. 7B ). In addition, ILTV replication was reduced by approximately 79% following PF573228 treatment (Fig. 7C, bottom) , as assayed by qPCR when all the ILTV-infected cells were dead (Fig. 7C, top) . Treatment with PF573228 in ovo, which did not induce embryo death by itself but which killed embryos in combination with ILTV infection during the observation period (data not shown), significantly reduced the level of virus production in allantoic fluid (Fig. 7D) and CAMs (Fig. 7E) . Histopathological examinations of slides with liver tissue stained with H&E showed more severe hepatic tissue necrosis in inoculated livers upon cotreatment with PF5732228 (with ILTV infection only, tissue necrosis was absent in four inoculated livers and mild in two; with ILTV infection and PF5732228 treatment, tissue necrosis was found to be mild in one inoculated liver, moderate in three inoculated livers, and severe in two inoculated livers) (Fig. 7F) . Correspondingly, a significant reduction of virus replication was observed in embryos inoculated with PF5732228 (Fig. 7G) . Collectively, our in ovo and in vitro data presented above support the notion that FAK activation is indispensable for Src regulation of ILTV infection.

Taken together and consistent with our hypotheses, our results demonstrate that Src and FAK interact with each other and play a critical role in controlling ILTV virulence and replication in vitro and in ovo.

Targeting the host molecular network that governs the responses of host cells to ILTV infection is a promising strategy for controlling ILT in a way that is independent of host immunity. The underlying mechanisms governing ILTV-host interactions largely remain unclear, but they are important for developing novel strategies to control ILT. In the current study, we identified the Src-FAK interaction to be an essential determinant of ILTV-associated cell death in LMH cells. Based on our findings, we suggest a model where, upon ILTV infection, Src and FAK are phosphorylated and form a positive-feedback loop, thus subsequently re-ducing ILTV-induced host cell death but maintaining ILTV replication at high levels in host cells. However, in cells where either Src or FAK is inhibited, the phosphorylation of Src and FAK is disrupted, thereby disrupting the Src-FAK positive-feedback loop. This has the subsequent effect of enhancing the virulence of ILTV by making host cells more susceptible to ILTV-induced cell death, but it also limits ILTV replication. Furthermore, our in ovo studies revealed that Src inhibition induced death only in heavily infected embryos and not in embryos with mild infections, although significant reductions in the levels of viral replication were obtained in both cases (Fig. 5) . We speculate that in chickens with minor ILTV infections, Src or FAK inhibition can help to quickly clear infected cells and delay disease progression, thereby limiting pathological damage. In turn, this might provide sufficient time for the activation of a host anti-ILTV immune response, thus improving the clearance of residual infected cells. However, in chickens heavily infected with ILTV, the inhibition of host Src or FAK could exacerbate the pathological effects and even increase the likelihood of death, although viral replication might still be repressed. Further investigations of the molecular basis of the regulation of ILTV replication and ILTV-induced cell death by the Src-FAK interaction are required to develop strategies that can improve ILT control and reduce economic losses during therapy.

Src was the first oncogene identified in the Rous sarcoma virus (RSV), which infects chickens (19) , and it has been implicated in multiple signaling pathways that regulate processes such as proliferation, survival, death, and angiogenesis (20) (21) (22) (23) (24) . Src is considered to be both a survival factor (32, 33) and a death factor (33) (34) (35) ; however, to the best of our knowledge, the effects of host Src on host cell death in human or animal herpesvirus infections have never been reported. The findings from the present study suggest that Src contributes to the survival of host cells in response to infection with gallid herpesvirus 1 but that it is not absolutely required for virus-induced cell death (Fig. 4A to D) .

In this study, we found that ILTV infection significantly promoted Src transcription but had a mild effect on the Src protein level at 2 dpi. Considering that only 10% of cells were positive for ILTV-gI staining at this time point, the difference in the expression pattern between the Src transcript and protein may be due to delayed translation of the Src transcript. We cannot rule out the possibility that oscillation of the Src mRNA/protein level or any posttranscriptional mechanism delays or even compromises the translation of additional Src transcripts. Actually, the increase in the amount of Src protein caused by ILTV infection may not be absolutely required for Src to modulate ILTV infection, because the phosphorylation of Src at Try 416, which maintains Src in an active conformation, is highly induced after ILTV infection, regardless of the absolute level of Src protein.

The interaction between Src and FAK has been shown to promote the entry of human herpes simplex virus 1 (HSV-1) into target cells (30, 31) . It is possible that Src-FAK cooperation has a similar effect on the cellular entry of ILTV. This assumption is based on the observation that the Src (51) and FAK (52) protein sequences are highly conserved (Ͼ95%) among humans, mice, and chickens. It has also been shown that HSV-1 VP11/12 can directly activate a host Src family kinase (53, 54) , while glycoproteins B, G, and H can activate it indirectly (55, 56) . Immunoprecipitation of the Src/FAK complex, followed by proteomic sequencing in LMH cells infected with ILTV, is needed to directly identify viral proteins that interact with Src/FAK. The ability of these candidate viral proteins to activate Src/FAK can be validated by infecting LMH cells using an ILTV harboring deletions in these genes. Further integrated, genome-wide high-throughput studies combined with a functional analysis of certain host factors may be helpful in exploring the mechanism by which ILTV hijacks host Src/FAK signaling in chicken cells.

It has been well documented in humans and mice that FAK and Src function mutually in integrin-mediated signaling: autophosphorylation of FAK at Tyr 397, which is stimulated by integrin, enables FAK to bind to the SH2 domain of Src, thereby resulting in the phosphorylation of Src at Tyr 416. The phosphorylation of Src at Tyr 416 in turn promotes the phosphorylation of FAK at Tyr 576 and Tyr 577 (57) (58) (59) (60) . To uncover the interaction between Src and FAK activation in our chicken model, the phosphorylation of Src at Tyr 416 and all three related FAK tyrosine residues was detected in detail. Consistent with the prior knowledge of Src and FAK in humans and mice, the phosphorylation of FAK at Tyr 576 and Tyr 577 upon ILTV infection in our model was dependent on Src activation ( Fig. 6B and C) , and the phosphorylation of Src at Tyr 416 by ILTV infection also required the phosphorylation of FAK (Fig. 6A) . However, the contribution of the phosphorylation of FAK at Tyr 397 to Src activation by ILTV in LMH cells is still unclear, because FAK is constitutively phosphorylated at Tyr 397 and is unaffected by ILTV infection (Fig. 6A) . The basal activity of Src is also essential for the maintenance of the constitutive phosphorylation of FAK at Tyr 397 (Fig. 6A ). Further investigation of the basal level of phosphorylation of FAK at Tyr 397, as well as its contribution to Src activation by ILTV in chicken cells in which FAK Tyr 397 is mutated, is required to answer this question.

Although Src activity may differ between the tumor cell line LMH and primary cells, considering the instability of the biological characteristics of different isolates and the short life span of primary cells during in vitro culture, the LMH cell line was chosen in the present study to establish a stable, experimental model for microarray assays and subsequent mechanistic studies. Given that the key host determinant of ILTV infection in the tumor cell line LMH is a well-known oncogene, it was crucial to determine whether the host Src/FAK-mediated mechanism that we found in LMH cells is also present in normal cells, especially in vivo. Hence, we first examined the effect of an Src/FAK inhibitor on ILTV replication in SPF embryos and observed an effect similar to that caused by the Src/FAK inhibitor in LMH cells ( Fig. 4 and 7) . Furthermore, during the in ovo study, we examined the histopathology of the livers of ILTV-infected chicken embryos and assayed the amount of virus transmitted to the liver. All the results support the notion that, instead of a tumor cell line-specific event, the mechanism that we identified in LMH cells is universal in normal cells and tissues. In addition, we also analyzed the only publicly available, genome-wide gene expression data for chicken embryo lung cells infected with a different virulent ILTV strain at an MOI of 0.1 (18) , which again revealed that host Src is the central regulator of the molecular network induced by ILTV infection in host cells (see Fig. S4 in the supplemental material).

Vaccination using live virus is the primary method by which ILT is prevented (6) . However, latent infections can still lead to outbreaks when the host immune system is compromised, such as under stress and at the onset of egg laying (13, 14, 61, 62) . Therefore, novel strategies that are independent of host immunity are required to successfully control ILT. For example, the administration of therapeutic agents that target the key determinants and signaling pathways that govern the interaction between ILTV and the host at the onset of infection could reduce the extensive use of live virus-based vaccines. Here, we have uncovered a key determinant of avian ILTV infection, thereby advancing our understanding of the ILTV-host interaction. Our results also support the notion that manipulating the host molecular response can alter the biological outcomes of ILTV infection. 

Increased virulence of modified live infectious laryngotracheitis vaccine virus following bird-to-bird passage

The effect of serial in vivo passage on the expression of virulence and DNA stability of an infectious laryngotracheitis virus strain of low virulence

Infectious laryngotracheitis vaccine virus detection in water lines and effectiveness of sanitizers for inactivating the virus

Detection of infectious laryngotracheitis virus from darkling beetles and their immature stage (lesser mealworms) by quantitative polymerase chain reaction and virus isolation

Immune responses to infectious laryngotracheitis virus

Infectious laryngotracheitis virus in chickens

Recombination in alphaherpesviruses

Characterization of infectious laryngotracheitis virus isolates from the US by polymerase chain reaction and restriction fragment length polymorphism of multiple genome regions

Characterization of infectious laryngotracheitis virus (ILTV) isolates from commercial poultry by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP)

Characterization of western European field isolates and vaccine strains of avian infectious laryngotracheitis virus by restriction fragment length polymorphism and sequence analysis

Epidemiology of recent outbreaks of infectious laryngotracheitis in poultry in Australia

Molecular epidemiology of infectious laryngotracheitis: a review

Effects of certain stress factors on the re-excretion of infectious laryngotracheitis virus from latently infected carrier birds

Latency and reactivation of infectious laryngotracheitis vaccine virus

Laryngotracheitis herpesvirus infection in the chicken: the role of humoral antibody in immunity to a graded challenge infection

The role of mucosal antibody in immunity to infectious laryngotracheitis virus in chicken

The role of cell-mediated immunity in chickens inoculated with the cellassociated vaccine of attenuated infectious laryngotracheitis virus

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src)

Signalling by Src family kinases: lessons learnt from DNA tumour viruses

Src family kinases, key regulators of signal transduction

Src family kinases: regulation of their activities, levels and identification of new pathways

Role of Abl and Src family kinases in actin-cytoskeletal rearrangements induced by the Helicobacter pylori CagA protein

SRC-family tyrosine kinases in oogenesis, oocyte maturation and fertilization: an evolutionary perspective

Focal adhesion kinase in integrin signaling

Suppression of anoikis in human intestinal epithelial cells: differentiation state-selective roles of ␣2␤1, ␣3␤1, ␣5␤1, and ␣6␤4 integrins

Integrin-regulated FAK-Src signaling in normal and cancer cells

Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein

Focal adhesion kinase and endothelial cell apoptosis

Focal adhesion kinase plays a pivotal role in herpes simplex virus entry

Focal adhesion kinase is critical for entry of Kaposi's sarcomaassociated herpesvirus into target cells

Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells

Dual role of Src kinase in governing neuronal survival

Inhibition of c-Src blocks oestrogen-induced apoptosis and restores oestrogen-stimulated growth in long-term oestrogendeprived breast cancer cells

c-Src modulates estrogen-induced stress and apoptosis in estrogen-deprived breast cancer cells

Infectious bronchitis virus nucleoprotein specific CTL response is generated prior to serum IgG

Complete genome sequence of the first Chinese virulent infectious laryngotracheitis virus

Detection of infectious laryngotracheitis virus by real-time PCR in naturally and experimentally infected chickens

Standardization of the methods and reference materials used to assess virus content in varicella vaccines

Monte Carlo simulation of the Spearman-Kaerber TCID50

Pharmacological activation of p53 triggers anticancer innate immune response through induction of ULBP2

Integrated high throughput analysis identifies Sp1 as a crucial determinant of p53-mediated apoptosis

Transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis virus infection

Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources

STRING v9.1: protein-protein interaction networks, with increased coverage and integration

Cytoscape: a software environment for integrated models of biomolecular interaction networks

SU6656, a selective Src family kinase inhibitor, used to probe growth factor signaling

Adhesion signaling-crosstalk between integrins, Src and Rho

Focal adhesion kinase inhibitors are potent anti-angiogenic agents

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src

Human T and B lymphocytes express a structurally conserved focal adhesion kinase, pp125FAK

Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes

Herpes simplex virus requires VP11/12 to activate Src family kinase-phosphoinositide 3-kinase-Akt signaling

Herpes simplex virus enhances chemokine function through modulation of receptor trafficking and oligomerization

Binding of herpes simplex virus type-1 virions leads to the induction of intracellular signalling in the absence of virus entry

1992. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain

Integrinmediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase

Laryngotracheitis (gallid-1) herpesvirus infection in the chicken. 4. Latency establishment by wild and vaccine strains of ILT virus

Replication and transmission of live attenuated infectious laryngotracheitis virus (ILTV) vaccines