A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics

Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator

Coordination of Rho GTPase activities during cell protrusion

The GTPases Rac1, RhoA and Cdc42 act in concert to control cytoskeleton dynamics1-3. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells4-7 and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac18, and Rac1 and RhoA are mutually inhibitory9-12. However, their spatiotemporal coordination, at the seconds and single micron dimensions typical of individual protrusion events, remains unknown. Here, we examine GTPase coordination both through simultaneous visualization of two GTPase biosensors and using a “computational multiplexing” approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 sec. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA plays a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions