Amino-terminal region of poliovirus 2C protein is sufficient for membrane binding.
The poliovirus-encoded, membrane associated polypeptide 2C is required for viral replication. We have previously established that, while the 2C protein lacks a defined membrane binding domain, the N-terminal region containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In order to determine whether the N-terminal region is sufficient for membrane binding, we have made fusion constructs between this region of 2C (amino acids 1-72 and 1-88) and a soluble protein, chloramphenicol acetyltransferase (CAT). The ability of CAT and the fusion polypeptides to bind to membranes was examined by in vitro translation in the presence of microsomal membrane. While CAT was found in the soluble fraction, both 2C/CAT fusion constructs (1-72/CAT and 1-88/CAT) were membrane associated, suggesting that the N-terminal region of 2C was sufficient to impart membrane binding. To confirm these results in vivo, CAT, 1-72/CAT, and 1-88/CAT were expressed in HeLa cells and their localization was examined using indirect immunofluorescence. Results presented here demonstrate that, while CAT is expressed throughout the cell, 1-72/CAT and 1-88/CAT constructs are capable of localizing to the endoplasmic reticulum (ER) area in transfected cells in the absence of other poliovirus proteins. These results suggest that the first 72 amino acids of 2C contain a membrane binding domain that is capable of targeting soluble proteins to the ER region of the cell.
Department of Microbiology and Immunology, The University of California, Los Angeles, School of Medicine, 90095, USA.
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Echeverri
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Banerjee
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Dasgupta
D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion; D017955:Poliovirus / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D017361:Viral Nonstructural Proteins; C076023:2C protein, viral