title: Identification of a region within the cytoplasmic domain of the subtype B Vpu protein of human immunodeficiency virus type 1 (HIV-1) that is responsible for retention in the golgi complex and its absence in the Vpu protein from a subtype C HIV-1.
abstract: The structure of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) is composed of a short Nterminal domain (NTD), a transmembrane domain (TM), and a cytoplasmic domain (CD). Previous studies have shown that the Vpu protein from subtype B HIV-1 is transported predominantly to the rough endoplasmic reticulum (RER)/Golgi complex compartments of the cell and is not incorporated into virions. Using a previously described VpuEGFP reporter system in which the Vpu protein was fused to the gene for enhanced green fluorescent protein (EGFP), we showed that the subtype B Vpu fusion protein was localized to the RER/Golgi region of the cell, similar to the native protein. In the present study, we show that fusion of the subtype C Vpu to EGFP results in a fusion protein that is transported to the cell surface. Using this reporter system, chimeric Vpu proteins in which the CD of the subtype B and C proteins were exchanged showed that the CD was sufficient for targeting the subtype B protein to the Golgi complex of the cell. Following identification of the cytoplasmic domain as being responsible for intracellular targeting, we then generated a series of mutants in which 13, 23, 31, 38, 51, and 56 amino acids were deleted from the cytoplasmic domain of subtype B Vpu. These deletion mutants were analyzed by SDS-PAGE for size, for membrane localization, and intracellular localization by confocal fluorescence microscopy. Our results indicate that the mutant with the carboxyl-terminal 13 amino acids deleted was still localized to the Golgi complex but mutants with 23, 31, 38, 51, and 56 amino acids from the carboxyl-terminus of the subtype B Vpu were transported to the cell surface. These results suggest that a signal for the retention of the subtype B Vpu within the Golgi complex resides in the second alpha-helical domain.
authors.affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Pacyniak
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Gomez
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Gomez
authors.forename: Ellyn R
authors.initials: ER
authors.lastname: Mulcahy
authors.forename: Mollie
authors.initials: M
authors.lastname: Jackson
authors.forename: David R
authors.initials: DR
authors.lastname: Hout
authors.forename: Billie J
authors.initials: BJ
authors.lastname: Wisdom
authors.forename: Edward B
authors.initials: EB
authors.lastname: Stephens
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D049452:Green Fluorescent Proteins; D054298:Human Immunodeficiency Virus Proteins; D011993:Recombinant Fusion Proteins; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins