Fig 2: Model of how cell-impermeable kinase inhibitors block viral entry.a Binding of HSV to cellular receptors (1) triggers intracellular calcium transients (2), which activate phospholipid scramblase-1 (3) leading to the translocation of phospatidylserines and cellular proteins (PDPK1, Akt, PLC? and possibly others) to the outer leaflet of the plasma membrane (4) where, analogous to the the canonical cytoplasmic signaling pathways, autophosphorylation of PDPK1 triggers phosphorylation of outer leaflet Akt, which, in turn, phosphorylates PLC? (5). This signaling pathway is required for HSV entry and is associated with subsequent restoration of phospholipid distribution (6). b Cell-impermeable kinase inhibitors (e.g. CIMSS) or antibodies to Akt block the activation of this extracellular signaling pathway (5) and prevent HSV entry resulting in (6) persistence of extracellular PtdS, which may lead to apoptosis.

Fig 3: Silencing of PDPK1, PLC?1, and Akt inhibits VSV-S infection.a Vero cells were transfected with control siRNA or siRNA targeting Akt1, PDPK1, PLC?, or FIC-1 and after 72 h, cell lysates were assayed by preparing western blots and probing for respective proteins. Cell lysates were probed for ß-actin on separate blots. Blots were scanned and and the percent reduction in protein expression of the silenced protein relative to siControl-transfected cells is indicated; blots are representative of two independent experiments. b Silenced cells were infected in duplicate with VSV-G or VSV-S and plaques quantified after 48 h incubation. Results are presented as mean ± SD and asterisks indicate significance relative to plaques formed on siControl wells (ANOVA, ****p < 0.0001). c Vero cells were transfected with siPDPK1 or control siRNA as in a and then infected with VSV-G or VSV-S. Following incubation for 30, 60, or 120 min, cells were fixed without or with Triton X-100 permeabilization and stained with antibodies to pAktT308 (red) or PDPK1 (green); nuclei were stained with DAPI. Images (Leica SP8, objective 63 × 1.4) are representative of results obtained in two independent experiments (bar = 10 µm). d Vero cells were infected with VSV-S or VSV-G in the presence of antibodies that target Akt (rabbit polyclonal), PDPK1 (rabbit polyclonal), the ACE2 receptor (murine monoclonal), or an murine monoclonal isotype control for 1 h, washed, and infection monitored by counting plaques 24 h pi. Results are presented as mean ± SD and asterisks indicate significance relative to plaques formed on isotype control antibody treated wells (**p < 0.01, ***p < 0.001, ****p < 0001).

Fig 4: Schematic illustration showing activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and how PRKAR1B-AS2 regulates it. Specific growth factor (GF) binds to its cognate receptor tyrosine kinase (RTK) leading to autophosphorylation of the tyrosine kinase domain and recruitment of PI3K to the inner layer of cell membrane. The catalytic subunit of PI3K, in turn, induces phosphorylation of phosphatidylinositol bisphosphate (PIP2) to produce the second messenger phosphatidylinositol triphosphate (PIP3). PIP3 and phosphoinositide-dependent kinase 1 (PDK1) activate protein kinase B (AKT) via phosphorylation at threonine residue (T308), resulting in sequential phosphorylation and activation of the downstream targets mammalian target of rapamycin (mTOR), S6 kinase (S6K), S6 ribosomal protein (rpS6), and 4E-binding protein 1 (4E-BP1). Activation of this pathway stimulates protein synthesis and proliferation and confers chemoresistance. PRKAR1B-AS2 induces PI3K-110a expression and phosphorylation of PDK1, AKT, and mTOR and therefore activating proliferation and conferring cisplatin and alpelisib resistance.

Fig 5: PDPK1 is phosphorylated upstream and PLC? downstream of Akt phosphorylation at the outer leaflet of the plasma membrane.a HaCat cells were transfected with siControl, siAkt1 or siPDPK1 and silencing assessed by preparing Western blots after 72 h and probing for Akt, PDPK1 and ß-actin; blots are representative of two independent experiments and relative Akt and PDPK1 expression is quantified after scanning images. b The siRNA-transfected cells were exposed to HSV-2(G) (MOI = 10 PFU/cell) (72 h after transfection) and at the indicated times post-infection (0, 30, or 120 min), the cells were fixed and stained (with or without Triton X-100 permeabilization) with antibodies to detect total (green) or phosphorylated AktT308 (red), total (green) or phosphorylated PDPK1S241 (red) or pPLC?1Y783 (red); nuclei were stained blue with DAPI. Images are representative of 2 independent experiments (Leica SP8, objective 63 × 1.4, bar = 10 µm). c The siRNA-transfected cells were infected with HSV-2(G) (100-200 PFU/well) and plaques were counted at 48 h; results are presented for two independent experiments each performed in duplicate as percent of PFU/well detected in the siControl transfected cells. The asterisks indicate p < 0.0001, unpaired t-test. d HaCat cells were infected with HSV-2(G) (MOI = 10 PFU/cell) in the presence of 10 U/ml of apyrase or control media and at the indicated times post-infection, fixed and stained (without permeabilization) with antibodies to detect phosphatidylserines (PtdS) and phospholipid scramblase (PLSCR1), pAktT308 or pPDPK1S241. Images (Leica SP8, objective 63 × 1.4, bar = 10 µm) are representative fields from two independent experiments.