Structural and functional analysis of the membrane-spanning domain of the human immunodeficiency virus type 1 Vpu protein.
The human immunodeficiency virus type 1 (HIV-1) vpu gene product is a class I integral membrane phosphoprotein that is capable of oligomerization. Two distinct biological activities have been attributed to Vpu: induction of CD4 degradation in the endoplasmic reticulum and enhancement of viral particle release from the plasma membrane of infected cells. These two biological activities were shown to involve two separable structural domains: the N-terminal transmembrane (TM) domain and the C-terminal cytoplasmic domain. The TM domain mediates enhancement of viral particle release, whereas phosphorylation of the cytoplasmic domain is essential for Vpu-induced CD4 degradation. In this study, we performed a mutational analysis of the TM domain of Vpu to delineate amino acids that are important in the process of viral particle release or in Vpu-induced CD4 degradation. Substitution of conserved amino acids from the N-terminal, middle, or C-terminal parts of the native VpuTM domain generated proteins that integrated normally into canine pancreatic microsomal membranes, exhibited subcellular localization similar to those of wild-type Vpu, but partially lost their ability to enhance viral particle release, strongly suggesting that the VpuTM domain contains determinants responsible for Vpu-mediated enhancement of viral particle release. Interestingly, the C-terminal TM mutant VpuIVW, in contrast to the other mutants, also lost its ability to bind and consequently degrade the CD4 molecule, indicating that the alteration of the C-terminal part of the TM did interfere with this function of Vpu. Taken together, our study supports the notion that both structural elements of Vpu (TM and cytoplasmic) contribute to the biological activities of Vpu.
Tiganos
E
E
Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, CP 6128, Succursale Centre-ville, Montréal, Québec, H3C 3J7, Canada.
Friborg
J
J
Allain
B
B
Daniel
N G
NG
Yao
X J
XJ
Cohen
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D005455:Fluorescent Antibody Technique; D015497:HIV-1 / Q000378:metabolism*; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D015531:Radioimmunoprecipitation Assay; D014158:Transcription, Genetic; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
D015704:CD4 Antigens; D054298:Human Immunodeficiency Virus Proteins; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins