Membrane cell fusion activity of the vaccinia virus A17-A27 protein complex.
Vaccinia virus enters cells by endocytosis and via a membrane fusion mechanism mediated by viral envelope protein complexes. While several proteins have been implicated in the entry/fusion event, there is no direct proof for fusogenic activity of any viral protein in heterologous systems. Transient coexpression of A17 and A27 in mammalian cells led to syncytia formation in a pH-dependent manner, as ascertained by confocal fluorescent immunomicroscopy. The pH-dependent fusion activity was identified to reside in A17 amino-terminal ectodomain after overexpression in insect cells using recombinant baculoviruses. Through the use of A17 ectodomain deletion mutants, it was found that the domain important for fusion spanned between residues 18 and 34. To further characterize A17-A27 fusion activity in mammalian cells, 293T cell lines stably expressing A17, A27 or coexpressing both proteins were generated using lentivectors. A27 was exposed on the cell surface only when A17 was coexpressed. In addition, pH-dependent fusion activity was functionally demonstrated in mammalian cells by cytoplasmic transfer of fluorescent proteins, only when A17 and A27 were coexpressed. Bioinformatic tools were used to compare the putative A17-A27 protein complex with well-characterized fusion proteins. Finally, all experimental evidence was integrated into a working model for A17-A27-induced pH-dependent cell-to-cell fusion.
Kochan
Grazyna
G
Department of Molecular and Cell Biology, Centro Nacional de Biotecnologia, CSIC, Madrid, Spain.
Escors
David
D
González
José Manuel
JM
Casasnovas
Jose Maria
JM
Esteban
Mariano
M
D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002522:Chlorocebus aethiops; D015726:Giant Cells / Q000821:virology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion; D013194:Staining and Labeling / Q000379:methods; D014616:Vaccinia virus / Q000235:genetics / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology*; D014760:Viral Fusion Proteins / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000502:physiology*; D053586:Virus Internalization; D000097573:Red Fluorescent Protein
D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C081257:A17L protein, Vaccinia virus; C526377:A27 protein, vaccinia virus