TNK2/ACK1 Strengthen Inuenza A Virus Infection by Blocking Viral Matrix 2 Protein(M2) into Lysosome to Degradation

TNK2/ACK1, a non-receptor tyrosine kinase, plays critical roles in signalling transduces and tracking. Our previous genome-wide CRISPR/CAS9 knockout screen revealed that mutant of TNK2 produced more restrict to inuenza virus infection. In this study, we aim to illustrate the role of TNK2 for inuenza A virus (IAV) replication in human cells.


Abstract
Background TNK2/ACK1, a non-receptor tyrosine kinase, plays critical roles in signalling transduces and tra cking.
Our previous genome-wide CRISPR/CAS9 knockout screen revealed that mutant of TNK2 produced more restrict to in uenza virus infection. In this study, we aim to illustrate the role of TNK2 for in uenza A virus (IAV) replication in human cells.

Results
CRISPR/Cas9-mediated mutant of TNK2 resulted in a signi cant reduction in viral proteins expression and viral titres for multiple in uenza strains, and furthermore, a decrease of nuclear import of IAV in the infected TNK2 mutant cells was observed in 3h post-infection. Interestingly, TNK2 mutation enhanced the colocalization of LC3 with autophagic receptor p62 and led to the attenuation of in uenza virus-caused accumulation of autophagosomes in TNK2 mutant cells. Further, confocal microscopy visualization result showed that in uenza viral matrix 2 (M2) was colocalized with Lamp1 in the infected TNK2 mutant cells in early infection, while almost no colocalization between M2 and Lamp1 was observed in IAVinfected wild-type cells. Moreover, TNK2 depletion also affected the tra cking of early endosome and the movement of in uenza viral NP and M2.

Conclusions
Our results identi ed TNK2 as a critical host factor for in uenza viral M2 protein tra cking, suggesting that TNK2 will be an attractive target for the development of antivirals therapeutics.

Background
In uenza caused by IAV is a severe respiratory disease capable of causing epidemics and public health security as well as economic loss. IAV is an enveloped RNA virus that contains negative-stranded RNA genome. IAV entry into host cells via receptor-mediated endocytosis. Endocytosed viruses are then tra cked into endosomes, where the low pH environment triggers the fusion of the viral and endosomal membranes, leading to viral genomes release 1,2,3,4 . In addition, during the viral life cycle, various of host factors are required by the virus to complete these processes 5 . Because IAV infection causes substantial morbidity and mortality, threatening public health as well as signi cant economic losses 6,7 , it is very important to nd e cient strategies to control virus infection and novel viral strain production.
The 2009 in uenza pandemic gave us some warnings that novel antiviral strategy needs to be created to improve viral clearance and the prevalence of pneumonia, reduce secondary bacterial infections. Current vaccines and antivirals directed targeting in uenza virus proteins have been developed and available to prevent annual epidemics 8,9 . However, IAV with genomic instability can rapidly develop resistance to these vaccines or antiviral drugs such as adamantanes, leading to ine cient protection against virus infection 10 . Combing with the limited number of viral drug directly targeting viral proteins and viral similar entry routes for replication, the development of new in uenza therapies targeting cellular factors required for viral replication will be of great attractive 9,11,12 . Multiple studies have been reported toward identifying host factors instead of virus proteins as drug targets by genome-wide screening approaches, including overexpression, arrayed or pooled RNAi screen, proteomic and CRISPR/Cas9 knockout or activated screen 13,14,15,16,17 .
TNK2 (activated Cdc42-associated kinase 1 or ACK1) is a multi-domain structural non-receptor tyrosine kinase, consisting of the Sterile alpha motif (SAM) domain, tyrosine kinase catalytic domain, a SH3 domain, GTPase binding domain (also known as Cdc42-binding domain), Clathrin interacting region, EGFR binding domain and an ubiquitin-association domain, leading to its functional complexity 18,19,20,21,22 . With its multi-structures, TNK2 is activated by multiple cellular signals and exploit various biological function though switching to different modes of kinase activation, resulting in adapt rapidly to cellular requirements 23 . In addition, TNK2 acts as an intermediary kinase that bridges the receptor tyrosine kinases (RTKs) and effecter proteins to control host cellular signalling transduces 24 .
Recently, Sylwia Jones reported that TNK2 interacts and colocalises with autophagic receptors p62/SQSTM1, leading to activated EGFR into autophagic degradative pathway, whereas silencing of TNK2 resulted in an increased location of EGFR in lysosome 25 . Previous studies showed that inhibition of tyrosine kinase activity or Receptor tyrosine kinase inhibitors leads to reduced virus uptake and progeny virus titers 26,27 , indicating that TNK2 may involve in virus replication by regulating the tra cking of receptor tyrosine kinase. Although there are no direct evidence illustrating the role of TNK2 in virus infection, a forward genetic screen showed that the Caenorhabditis elegans ortholog of TNK2, sid-3 has been identify as host factors critical for Orsay virus infection 28 . Moreover, multiple genome-wide RNAi screens also revealed that TNK2 can act as a potential candidate involved in virus infection, including in uenza A virus (IAV), hepatitis C virus (HCV), and vesicular stomatitis virus (VSV) 13,29,30,31,32,33 .
Collectively, these data indicate that TNK2 may participate in IAV infection, although the function of TNK2 remains unanswered.
In this study, we analysed the role of TNK2 for IAV infection. We found that CRISPR/Cas9-meidiated mutant of TNK2 reduced the viral replication and destroyed IAV infection-induced accumulation of autophagosomes. Further studies demonstrated that the mechanism by which TNK2 mutation enhanced the fusion of autophagosome with lysosomes was by mediating in uenza matrix protein 2 (M2) tra cking into the classical lysosomal pathway.

Results
Generation of TNK2 mutant single-colony by CRISPR/Cas9 technology In order to produce the indel mutant of TNK2, dual gRNAs were designed to delete the exon 2 (181bp) of TNK2 (ENSG00000061938) (Fig. 1A). By co-transfecting cells with Cas9 and dual TNK2 gRNAs vectors, drug-selectable A549 cells were established. BFP-positive cells were screened by supplementing the culture medium with 1.5 ug/ml puromycin for 10 days. after puromycin selection, the single colonies were picked and detected with molecular biology technology. PCR and Sanger sequencing revealed that TNK2-A549#15 and TNK2-A549#12 monoclonal is a homozygous mutation with an almost 660bp nucleotide deletion, respectively (Fig. 1A). In addition, Western blotting and qPCR analysis of these two mutant cells (TNK2-A549#15 and TNK2-A549#12) showed a signi cate reduced in mRNA and protein levels of TNK2 compared with wild-type (WT) ( Fig. 1B and 1C). qPCR results showed that TNK2 was almost undetectable in TNK2-A549#15 (TNK2-KO) cells. Taken together, TNK2 homozygous mutation clone was generated via CRISPR/Cas9 technology.

TNK2 de ciency inhibits different species of in uenza virus replication
To verify whether TNK2 is required for in uenza A virus infection, immuno uorescence staining for in uenza virus proteins NP as a marker of viral ribonucleoprotein (vRNP) localization and M2 as an ion channel protein were used to assess virus replication. TNK2-A549#15, TNK2-A549#12 and wild-type A549 cells were infected with human in uenza virus (MOI 10). After 3h post-infection, a strong NP signal was detected in the nuclei of infected cells, while in TNK2 knockout cells, barely any nuclear NP was detectable and most of NP seemed to attach with membrane ( Fig. 2A). In addition, knockout of TNK2 also reduced NP and M2 expression levels ( Fig. 2A, 2B), indicating that TNK2 knockout interferes dissociation of the viral RNA and blocks nuclear import of in uenza virus vRNPs. Moreover, the virus titer was strongly reduced in TNK2-KO cells compared to those for wild-type cells. Additionally, swine in uenza virus (HB, H1N1) and avian in uenza virus (CK, H9N2-GFP) also displayed reduced growth in A549 cells upon TNK2 knockout ( Fig. 2C, 2D, 2E), suggesting that the function of TNK2 in in uenza virus replication may be conserved.

TNK2 de ciency induced the formation of autolysosome during in uenza virus infection
Previous study has illustrated that in uenza virus infection inhibits autophagosome maturation to enhance viral budding and virion stability, and TNK2 is implicated in receptor endocytic tra cking and lysosome degradative pathway. In order to investigate whether the accumulation of autophagosomes caused by in uenza virus infection was altered under deletion of TNK2, western blotting analysis showed that the amount of endogenous lapidated Atg8/LC3-II form in in uenza-infected wild-type cell was signi cantly accumulated than those in in uenza-infected TNK2 knockout cells. Moreover, the expression of p62 protein, which is used to monitor autophagic ux, was strongly reduced in in uenza-infected TNK2 mutant cells compared to in uenza-infected wild-type cell (Fig. 3A), demonstrating that deletion of TNK2 is able to weaken the accumulation of autophagosomes induced by in uenza infection. In addition, to con rm this promotion of autophagosome fusion with lysosomes in TNK2 mutant cells infected with in uenza virus, we investigated the colocalization of LC3 with p62 and Lamp1 visualized by confocal microscopy, respectively (Fig. 3B, 3C). The results showed that p62 was overlapped with autophagosomes upon in uenza infection in both wild-type and TNK2-KO cells, but the greater number of overlapped dots were found in TNK2-KO cells. Additionally, LC3 was not found to co-localize with Lamp1 and the expression of Lamp1 was much lower in wild-type cells with in uenza virus infection, while this autophagosome marker LC3 was partially overlapped with Lamp1 in TNK2-KO cells with in uenza virus infection. These data suggested that TNK2 de ciency can promote the fuse of autophagosomes with lysosomes during in uenza virus infection.
TNK2 regulates the tra cking of in uenza virus M2 protein into lysosome degradative pathway To illustrate more precisely how TNK2 regulates autolysosome formation during in uenza virus infection, we rstly detected intracellular dynamic location of in uenza virus M2 in TNK2 knockout cells. The results showed that silencing of TNK2 resulted in the different localization of in uenza virus NP and M2 protein in early infection (Fig. 4). Then we investigated whether the tra cking of in uenza virus M2 is impaired in the absence of TNK2. As shown in Fig. 5A, in uenza virus M2 did colocalise with Lamp1 in in uenza virus infected TNK2-KO cells, while barely colocation singling was detected in wild-type cells. Reversely, the colocation of M2 with p62 was accumulated in wild-type cells, although there were more punctate p62-positive structures that did not localize with M2 in TNK2-KO cells (Fig. 5C). In addition, given that TNK2 partially localises to EEA1-positive compartment, where early endosomes are required for autophagosomal maturation, we also analysed whether the fusion of early endosomes with autophagosomes is affected in cells depleted of TNK2. After 1h post-infection, when compared with the wild-type cells, knockout of TNK2 triggered the colocalization of EEA1-postive early endosome with Lamp1-postive lysosome (Fig. 5B). Interestingly). Altogether, these data show that TNK2 associates with in uenza virus M2 tra cking and is involved in in uenza virus-induced autophagosome accumulation.
Overexpression of TNK2 rescues in uenza virus infection and blocks the degradation of EEA1-postitive early endosome in TNK2-knockout cells To assess whether TNK2 expression can rescue in uenza virus infection in TNK2-knockout cells, rescues assay was adopted to evaluate the biological effects of TNK2 overexpression on in uenza virus infection in TNK2-knockout cells. FLAG-tagged TNK2 vector was transfected into TNK2-knockoout cells, followed by infection with in uenza A/WSN/33 virus. Weston blotting assay showed that TNK2 overexpression increased the expression of viral protein NP, HA and M2 in TNK2-knockout cells (Fig. 6A), and Immuno uorescence assay also showed that TNK2 overexpression enhanced in uenza virus propagation in TNK2-knockout cells (Fig. 6B), determining that TNK2 restored proviral activity in the TNK2 knockout cells. Furthermore, the confocal microscopy showed that early endosome maker EEA1 no long colocalized with lysosome maker Lamp1 in TNK2 knockout cells (Fig. 6C), suggesting that TNK2 is critical for localization of early endosomes and escape of in uenza virus from early endosomes.

TNK2 inhibitors decrease in uenza virus replication
To investigated the antiviral activity of TNK2 inhibitor, two inhibitors (XMD8-87 and AIM-100) were used to assess the replication of in uenza virus. Weston blotting data showed that total TNK2 and phosphorylated TNK expressions were decreased after both XMD8-87 and AIM-100 treatment (Fig. 7A).
Subsequently, compared with control, western blotting results showed that both XMD8-87 and AIM-100 e ciently reduced in uenza viral M2 expression levels in dose dependant manner (Fig. 7B), suggesting that lower levels of TNK2 can e ciently protect host against in uenza virus infection.

Discussion
In the present study, we demonstrate a novel role for TNK2 in in uenza virus infection. Our data showed that TNK2 served as a pivotal regulator of in uenza virus and was required for the tra cking of virus protein and the accumulation of in uenza virus-induced autophagosomes. CRISPR/Cas9 mediated TNK2 de ciency appears to impair endosomal maturation, as supported by directly delivering early endosomes to Lamp1-positive lysosomes. Furthermore, we found that knockout of TNK2 triggered autophagic ux that was impaired in in uenza virus infection by degrading in uenza viral protein M2 by lysosome pathway. Moreover, we found that TNK2 inhibitors were also able to inhibit virus replication. This suggested that TNK2 played important roles in in uenza virus infection.
Early endosomes have a low pH that is very critical for the fusion of the viral and endosomal membranes and the opening of in uenza viral M2 involved in viral genomic release 36,37 . Our data showed that in uenza viral M2 exhibited a different localization pattern in TNK2 knockout cells comparing with wild type cells in in uenza virus infection. Notably, a large number of early endosome and viral M2 were found in Lamp1-positive lysosome in TNK2 knockout cells. Thus, we conclude that TNK2 may control early endosome-lysosome tra cking in viral infection. Interestingly, in uenza virus M2 protein could block autophagosome fusion with lysosomes by directly interacting with LCE and driving LC3 relocalization to plasma membrane 38,39 . Additionally, previous studies suggest that TNK2 localized to early endosomes and autophagosomes upon stimulation with EGF 25 . Therefore, we propose that TNK2 may be critical for in uenza virus M2 protein-induced autophagosome accumulation.
In addition, TNK2 acts as a major integrator in ligand-induced degradation of EGFR and suppression of the ACK1 expression by siRNA resulted in inhibition of EGF-induced degradation of EGFR 40 . Accordingly, EGFR knockdown also impaired e cient uptake of in uenza virus into A549 cells and in uenza virus. In addition, when in uenza virus infected host cells, the attachment of in uenza virus induced EGFP endocytosis and EGFR kinase activity required for in uenza virus internalization 41 . Because ACK1 contained a clathrin-binding motif, interacted with clathrin, and participated in clathrin-mediated endocytosis 42 . Thus, we hypothesize that ACK1 may control EGFR internalization and clathrin-mediated endocytosis to block the uptake of in uenza virus.

Conclusions
In summary, our studies identify a novel role of TNK2 in regulating in uenza virus infection. Our data indicate that TNK2 regulates in uenza virus M2 transport to effect autophagy ux and highlight TNK2 may be as a potential antiviral target against in uenza virus infection.

Cells and viruses
A549 cells were maintained in DMEM F-12 medium (Gibco). MDCK and 293T cells were maintained in Dulbecco's modi ed Eagle's medium (DMEM) (Invitrogen) with high glucose. All of the media was supplemented with 2 mM L-glutamine, 100 U ml penicillin/streptomycin and 10% foetal bovine serum (FBS, Life Technologies). All cells were cultured in a humidi ed incubator with 5% CO 2 at 37℃.
Generation of TNK2-KO-A549 Cell Line A549 cells were co-transfected with TNK2-KO-gRNA1 and TNK2-KO-gRNA2 using the Lipofectamine® 3000 reagent (Invitrogen) according to the manufacturer's instructions. At 24 h after transfection, cells were selected with 1.5 µg/mL of puromycin (Invitrogen) which was diluted in DMEM with 10% FBS. Seven days later, the positive clones were isolated, trypsinized and diluted in 96-well plates. The single cell clones were then further used to extract DNA by DNeasy Blood & Tissue Kit (Qiagen) and ampli cated by PCR with primers (Forward: TCCGTCACATCTAAGGAGCC and Reverse: GAGCACGAATCAGCAAACCA) and the PCR products were performed DNA sequencing analysis.

Virus infection
Cells were washed with PBS and then infected with in uenza at the indicated multiplicity of infection (MOI) in the infection buffer (PBS supplemented with 0.3% bovine serum albumin) for 60 min on ice. Cells were next washed twice with PBS and incubated in DMEM supplemented with 0.3% bovine serum albumin, with 2 mM L-glutamine, 100 U ml penicillin/streptomycin and 1 µg/ml TPCK-trypsin at 37°C. The samples were harvested at indicated postinfection for further analysis Immuno uorescence microscopy The colocalization between in uenza viruses and cellular early endosomes was assessed using confocal microscopy. Cells were grown on glass coverslips. The next day, cells were infected with A/WSN/33 virus at a MOI of 10 on ice for 1h and then were incubated for the indicated times at 37℃. Cells were xed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 and blocked with 2% bovine albumin. Cells were then incubated with primary antibodies overnight at 4℃. After the cells were incubated with primary antibodies, they were washed three times with PBS before uorescently labelled secondary antibodies (Abcam) were added for 1 to 2h at room temperature. Nuclei were stained with DAPI, and slides were mounted using ProLong Antifade (Invitrogen). All the specimens were analysed by a confocal laser-scanning microscope (Leica SP8) and images were acquired using the LAS X software.
For the immuno uorescence analysis of the endosome-to-TGN retrograde tra cking, wild type and TNK2-Knockout cells were grown on coverslips and were rstly incubated on ice for 30min before moving to the 37℃ incubators at the indicated times. Cells were xed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 and blocked with 2% bovine albumin. Cells were then incubated with primary antibodies overnight at 4℃. After the cells were incubated with primary antibodies, they were washed three times with PBS before uorescently labelled secondary antibodies were added for 1 to 2h at room temperature. Nuclei were stained with DAPI, and slides were mounted using ProLong Antifade (Invitrogen). All the specimens were analysed by a confocal laser-scanning microscope (Leica SP8) and images were acquired using the LAS X software. Western blots 3x10 6 cells were trypsinized and pelleted at 500g for 5 minutes. Cells were lysed with a RIPA buffer (25mM Tris•HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Invitrogen) at ice for 20 minutes. Protein concentration was measured using the Bradford assay (Pierce). 20µg protein per sample was loaded onto a 4-20% TGX Stain-Free Gel (Bio-Rad). Protein was transferred to a PVDF membrane at 200mA for 120 minutes. Blots were blocked overnight with 5% milk powder in PBS + 0.1% Tween20 before incubation with the primary antibodies at a 1:500 dilution in PBS + 0.1% Tween 20 for overnight. Blots were washed three times with PBS + 0.1% Tween 20, and probed with the HRP conjugated secondary antibodies at 1:5,000 dilution for 1 hour, then washed again for 3 times. The western blots were developed using the Clarity Western ECL Substrate (Bio-rad) and imaged with the Amersham Hyper lm ECL system (GE Healthcare). Primary antibodies used for this experiment included: Rabbit anti-COG8(GeneTex) antibody, Rabbit anti-GAPDH antibody (GeneTex), Rabbit anti-M2 antibody (GeneTex)., mouse anti-actin antibody (CST), Rabbit anti-HA antibody (GeneTex), rabbit anti-NS2 antibody (GeneTex), and mouse anti-NP antibody (Abcam). Goat anti-rabbit and goat anti-mouse secondary antibody linking with HRP were purchased from Invitrogen. All bands of western blots were detected within the linear range.
qRT-PCR Total RNA extracts from each sample were obtained using the Arcturus Picopure RNA Isolation Kit (Invitrogen) following the manufacturer's instructions, and then complementary DNA was synthesized using the Superscript III reverse transcriptase kit (Invitrogen). Real-time RT-PCR was performed using the SYBR Green Real Time PCR Master Mix (Toyobo Biologics) in the LightCycler 480 (Roche Molecular Biochemicals). Individual transcripts in each sample were assayed three times. The PCR conditions were as follows: initial denaturation for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C and 40 s at 72°C. Relative expression levels of gene expression was determined by evaluating the threshold cycle (Ct) of target gene after normalization against the Ct value of GAPDH (housekeeping gene) dependent on the delta delta cycles to threshold (ΔΔCT) method. Primers used for the qRT-PCR assays were: TNK2 (Forward: GCAAGAGGCGAATTGGCTG and Reverse: CCTTCCCGTTCAGGTAGGTT) and GAPDH (Forward: ACAACTTTGGTATCGTGGAA GG and Reverse: GCCATCACGCCACAGTTTC).

Statistical analyses
Data were expressed as means ± standard errors of the means (SEM). Statistical analysis was performed by paired two-tailed Student's t test. P value equal or lower to 0.05 was considered signi cant (*p < 0.05, **p < 0.01, ***p < 0.001).