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.

@article{citeulike:218144, 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.}, address = {Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160.}, author = {Pacyniak, E. and Gomez, M. L. and Gomez, L. M. and Mulcahy, E. R. and Jackson, M. and Hout, D. R. and Wisdom, B. J. and Stephens, E. B. }, citeulike-article-id = {218144}, doi = {10.1089/aid.2005.21.379}, issn = {0889-2229}, journal = {AIDS Res Hum Retroviruses}, keywords = {binding, gene, hiv, ic, new, site}, month = {May}, number = {5}, pages = {379--394}, posted-at = {2007-08-02 23:32:34}, priority = {2}, 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.}, url = {http://dx.doi.org/10.1089/aid.2005.21.379}, volume = {21}, year = {2005} }

TY - JOUR ID - citeulike:218144 L3 - citeulike-article-id:218144 TI - 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. JF - AIDS Res Hum Retroviruses VL - 21 IS - 5 SP - 379 EP - 394 AD - Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160. SN - 0889-2229 N2 - 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. KW - binding KW - gene KW - hiv KW - ic KW - new KW - site AU - Pacyniak, E AU - Gomez, ML AU - Gomez, LM AU - Mulcahy, ER AU - Jackson, M AU - Hout, DR AU - Wisdom, BJ AU - Stephens, EB PY - 2005/05// UR - http://dx.doi.org/10.1089/aid.2005.21.379 ER -