Genome-wide siRNA screening against adenovirus infection
Final Report Abstract
In the described siRNA screen targeting 50 cellular kinases, mTOR (mammalian target of rapamycin) was found to cause reduction of Ad5-GFP and Ad3-GFP infection upon knock down. Another kinase (adaptor-associated kinase 1; AAKl) was found to be involved exclusively in Ad5-GFP infection. mTOR (mammalian target of rapamycin) is a cellular kinase involved in a broad spectrum of biosynthetic and regulatory activities. It has also been implicated in DNA tumor virus infections. In higher eukaryotic cells it is found in two complexes, the rapamycin-sensitive mTORC1 and the caffeine-sensitive mTORC2. mTORC1 acts through the adaptor protein raptor and regulates cell growth and division. mTORC2 uses the adaptor protein rictor and regulates cell survival, membrane traffic and actin cytoskeleton dynamics. mTOR is also intensely studied in cancer research. Detailed knowledge about upstream and downstream signalling of mTOR is of fundamental interest to understand functions and malfunctions in disease. In particular for the therapy of cancer. We showed that mTORC1 and mTORC2 have distinct roles in Ad5 replication. While the knock-down of rictor reduced infection with Ad5-GFP as well as infection with Ad5 wt, the knock down of raptor had little effects on infection with Ad5-GFP but it inhibited the expression of the immediate early Ad5 wt transactivator protein E1A. Furthermore, the amount of progeny virius originating from Ad5 wt infected mTOR, rictor, or raptor knock down cells was reduced which also implicates a role of raptor in later steps of adenovirus replication. On the uptake of other ligands by clathrin mediated endocytosis, the knock-down of either mTOR or raptor strongly reduced the levels of transferrin and slightly those of low-density lipoproteins. Knock down of rictor had just minor effects. Further data imply a common pathway for mTOR and PKCa. As we could show stimulation of PKCs rescued the inhibition of infection that occurs in mTOR knock down cells. As shown before PKCa knock down leads to an accumulation of viral particles in endosomal compartments. In summary, these data arrive at three conclusions: 1. Both mTOR complexes are involved in adenovirus infection, whereas the rictor containing complex mTORC2 acts on early entry events like endocytosis, endosomal escape or intracellular transport and mTORC2 (raptor containing complex) plays a role later in replication. 2. mTORC2 might not act on clathrin-mediated endocytosis directly as other ligands are independent of this comoplex. It is more likely that mTORC acts on receptor induced signalling pathways. 3. mTOR and PKCa may act in concert on the endosomal escape of viral particles.
