Membrane-association properties of avian encephalomyelitis virus protein 3A.
Avian encephalomyelitis virus (AEV) protein 3A is a membrane-interacting protein containing a stretch of 21 hydrophobic amino acid residues. Membrane-association property was assayed using chick embryo brain (CEB) cells transfected with the fusion GFP-3A and its various deletion mutants demonstrate that 3A is integrally interacted with membranes by its hydrophobic domain and further defines that the motif of amino acid residues 45-51, the most C-terminal hydrophobic domain essential for this feature. Expression of 3A in transfected CEB cells results in membrane permeability modifications through association of the third motif with membranes, which can be demonstrated by release of lactate dehydrogenase (LDH) into the medium. Furthermore, the localization of the protein 3A in transfected CEB and Cos-7 cells exhibited an overlapping staining pattern with an endoplasmic reticulum (ER) and involved in the disassembly of the Golgi apparatus under double-staining and confocal microscopic observations, whereas the 3A mutants lacking amino acids 45-51 could not localize to the ER and display an intact Golgi morphology as seen in the mutant devoid of the complete hydrophobic domain after transfection. Taken together, our results demonstrate that the motif (aa 45-51) of the transmembrane domain might be fundamental for the stable interaction of the protein 3A with the ER membrane regardless of the cell types. Although this motif was deleted, the resultant protein did not localize to the ER, which directly results in the loss of the ability to block the ER-to-Golgi transport by 3A protein and hence makes the morphology of the Golgi apparatus return to normal.
Liu
Jue
J
Animal Health Biotechnology, Temasek Life Sciences Laboratory, The National University of Singapore, Singapore 117604, Singapore.
Wei
Ting
T
Kwang
Jimmy
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism / Q000473:pathology / Q000821:virology; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism / Q000473:pathology / Q000821:virology; D002478:Cells, Cultured; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D001350:Encephalomyelitis Virus, Avian / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008969:Molecular Sequence Data; D009336:Necrosis; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D017361:Viral Nonstructural Proteins