Amino terminal regions of poliovirus 2C protein mediate membrane binding.
The poliovirus-encoded, membrane-associated polypeptide 2C is required for viral replication. We have expressed 2C, its precursor 2BC, and a number of 2C deletion mutants in eukaryotic (HeLa) cells and examined their localization using indirect immunofluorescence. Results presented here demonstrate that proteins 2C and 2BC are capable of localizing to the endoplasmic reticulum area in transfected cells in the absence of other poliovirus proteins. Additionally, 2C binds tightly to microsomal membranes in a direct in vitro membrane binding assay. Although the 2C protein lacks a defined membrane binding domain, we demonstrate that the N-terminal region encompassing amino acids 21-54 and containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In contrast, most of the C-terminus portion of the protein appears to be unnecessary for membrane association. The susceptibility of membrane-associated 2C to proteinase K digestion in vitro suggests that all or most of 2C is exposed to the cytoplasmic face of the membrane. Furthermore, unlike most proteins targeted to the endoplasmic reticulum, 2C does not appear to be glycosylated or cleaved by signal peptidases. The implication of the membrane binding domain of 2C in viral RNA replication is discussed.
Echeverri
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AC
Department of Microbiology and Immunology, UCLA School of Medicine 90024, USA.
Dasgupta
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A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D017384:Sequence Deletion / Q000502:physiology; D014616:Vaccinia virus / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology
C091441:2BC protein, Poliovirus; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C076023:2C protein, viral