title: Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation.
abstract: Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.
authors.affiliation: Laboratory of Cellular Oncology, NCI, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Jobbagy
authors.forename: Terry H
authors.initials: TH
authors.lastname: Bird
authors.forename: Maribeth V
authors.initials: MV
authors.lastname: Eiden
authors.forename: Wayne B
authors.initials: WB
authors.lastname: Anderson
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002459:Cell Fusion; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry; D015686:Gene Products, env / Q000235:genetics; D005799:Genes, Dominant; D015726:Giant Cells / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000378:metabolism; D008561:Membrane Fusion; D051379:Mice; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry; D011493:Protein Kinase C / Q000502:physiology*; D051744:Protein Kinase C-epsilon; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D050608:Sodium-Phosphate Cotransporter Proteins; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D027981:Symporters / Q000502:physiology*; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D014759:Viral Envelope Proteins / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D000939:Epitopes; D015686:Gene Products, env; D010455:Peptides; D034741:RNA, Small Interfering; D050608:Sodium-Phosphate Cotransporter Proteins; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D027981:Symporters; D014759:Viral Envelope Proteins; D004247:DNA; D000242:Cyclic AMP; C497256:Prkce protein, mouse; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon