A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection

Abstract

Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrinindependent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.

Interaction Between Phosphoinositide 3-Kinase and the VDAC2 Channel Tethers Endosomes to Mitochondria and Promotes Endosome Maturation

“…To reveal the molecular mechanism by which the Ras-PI3K complex is translocated from the plasma membrane to endosomes in a RAPEL-dependent manner (Fujioka et al, 2019), we screened for RAPEL binding proteins with the use of a yeast two-hybrid assay based on the RBD of PI3K (PI3K-RBD) as the bait ( Figure S1A ). We identified six candidate proteins ( Table S1 ), among which we focused on the mitochondrial pore protein VDAC2 because we were able to confirm its interaction with RAPEL in mammalian cells by immunoprecipitation analysis ( Figure 1A ).…”

“…The expression vectors pCAGGS-VN-H-Ras-WT, pCXN2-FLAG-PI3K(p110γ)-RBD-VC, pFX-SECFP, pFX-ECFP-RAPEL, pFX-mito-SECFP, and pFX-mito-EGFP were described previously (Fujioka et al, 2018, 2019; Kashiwagi et al, 2019; Tsutsumi et al, 2009). The vector pCMV-TagRFP-EEA1 was obtained from Addgene (Cambridge, MA, USA).…”

“…Analysis of the endosomal localization of the Ras-PI3K complex was performed as described previously (Fujioka et al, 2019). In brief, confocal images of cells expressing Venus NH2–terminus-Ras (VN-H-Ras-WT), PI3K-RBD–Venus COOH-terminus (PI3K-RBD-VC), and TagRFP-EEA1 were acquired, and background signals were subtracted.…”

“…We have previously shown that endosomal localization of a complex formed by the small GTPase Ras and PI3K promotes clathrin-independent endocytosis and endosome maturation (Fujioka et al, 2011). In addition, a 28–amino acid sequence at the NH 2 -terminal region of the Ras binding domain (RBD) of PI3K was found to be required for such endosomal localization and was thus designated RAPEL ( R as- P I3K e ndosomal localization) (Fujioka et al, 2019). However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes in a RAPEL-dependent manner has remained to be investigated.…”

Folding latency of fluorescent proteins affects the mitochondrial localization of fusion proteins

“…These sequences were then subcloned into the XhoI/NotI sites of the pFX-CAAX vectors , which contains human cytomegalovirus (CMV) promoter (Fujioka et al, 2019).…”