Influenza 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 trafficked into endosomes, where the low pH environment triggers the fusion of the viral and endosomal membranes, leading to viral genomes release1,2,3,4. In addition, during the viral life cycle, various of host factors are required by the virus to complete these processes5. Because IAV infection causes substantial morbidity and mortality, threatening public health as well as significant economic losses 6,7, it is very important to find efficient strategies to control virus infection and novel viral strain production.
The 2009 influenza 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 influenza virus proteins have been developed and available to prevent annual epidemics8,9. However, IAV with genomic instability can rapidly develop resistance to these vaccines or antiviral drugs such as adamantanes, leading to inefficient protection against virus infection10. Combing with the limited number of viral drug directly targeting viral proteins and viral similar entry routes for replication, the development of new influenza therapies targeting cellular factors required for viral replication will be of great attractive9,11,12. 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 complexity18,19,20,21,22. 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 requirements23. In addition, TNK2 acts as an intermediary kinase that bridges the receptor tyrosine kinases (RTKs) and effecter proteins to control host cellular signalling transduces24. 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 lysosome25. Previous studies showed that inhibition of tyrosine kinase activity or Receptor tyrosine kinase inhibitors leads to reduced virus uptake and progeny virus titers26,27, indicating that TNK2 may involve in virus replication by regulating the trafficking 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 infection28. Moreover, multiple genome-wide RNAi screens also revealed that TNK2 can act as a potential candidate involved in virus infection, including influenza 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 influenza matrix protein 2 (M2) trafficking into the classical lysosomal pathway.