A Ca2+-dependent signalling circuit regulates influenza A virus internalization and infection

Abstract

Various viruses enter host cells via endocytosis, but the molecular mechanisms underlying the specific internalization pathways remain unclear. Here we show that influenza A viruses (IAVs) enter cells via redundant pathways of clathrin-mediated and clathrin-independent endocytosis, with intracellular Ca2+ having a central role in regulation of both pathways by activating a signalling axis comprising RhoA, Rho-kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and phospholipase C (PLC). IAV infection induces oscillations in the cytosolic Ca2+ concentration of host cells, the prevention of which markedly attenuates virus internalization and infection. The small GTPase RhoA is found both to function downstream of the virus-induced Ca2+ response and itself to induce Ca2+ oscillations in a manner dependent on Rho-kinase and subsequent PIP5K-PLC signalling. This signalling circuit regulates both clathrin-mediated and clathrin-independent endocytosis during virus infection and seems to constitute a key mechanism for regulation of IAV internalization and infection

Fluorescent Protein-based Biosensors to Visualize Signal Transduction beneath the Plasma Membrane

“…4B). 10 Furthermore, we also revealed that the virusdependent calcium elevation triggered RhoA activation only at the periphery of the plasma membrane, as visualized by RaichuRhoA (Fig. 5).…”

“…29 The region of RhoA activation was too restricted for the activation to be detected by conventional biochemical methods. 10 Imaging-based techniques might thereby maximize the researchers' ability to visualize such compartmentalized phenomena that have remained undiscovered by conventional approaches.…”

“…1B). 10 Recently, Cameleon was further improved by Horikawa and Nagai with respect to its measurable range of calcium concentrations by engineering the Ca 2+ -sensing domain of Cameleon. This biosensor, Cameleon-Nano, achieved a higher Ca 2+ affinity, which resulted in approximately a 50-fold reduction in its Kd value (to 15 nM) compared with the classical Cameleon protein.…”

“…3). 10 Because these achievements have depended to a large extent on live-cell imaging using fluorescent protein-based biosensors, we summarize herein the biosensors that are useful for visualizing signal transduction beneath the plasma membrane. …”

Interplay between influenza A virus and host factors: targets for antiviral intervention

“…A signaling pathway involving phosphatidylinositol 3-kinase (PI3K) have been demonstrated to be involved in virus entry by specifically mediating clathrin-independent endocytosis, endosomal maturation, and intracellular transport of viruses [104]. Recently, it has been shown that Ca ?2 plays an indispensable role in IAV internalization by regulating the phosphatidylinositol 5-kinase (PI5K) and phospholipase C (PLC) signaling pathways [105]. These pathways constitute key mechanisms for clathrin-dependent and clathrin-independent endocytosis during virus infection.…”