Functional analysis of the phosphorylation sites on the human immunodeficiency virus type 1 Vpu protein.
The human immunodeficiency virus type 1 (HIV-1)-encoded vpu product is a small class 1 integral membrane protein that is phosphorylated by the ubiquitous casein kinase II (CKII) in HIV-1-infected cells. The Vpu protein facilitates the release of budding virions from the surface of infected cells and delays the rate of syncytium formation. In this study, we investigated the role of phosphorylation in the biological activity of Vpu. Our results show that phosphorylation of Vpu occurs on serine residues at positions 52 and 56 located in a highly conserved dodecapeptide sequence. Mutation of either Ser 56, or both Ser 52 and Ser 56 impaired the ability of Vpu to delay the rate of syncytium formation while retaining virion release activity at levels comparable to vpu+ proviruses. Flow cytometry analysis indicates that the relative amounts of envelope glycoprotein gp120 expressed at the surface of cells transfected with these vpu mutant proviruses was two- to threefold greater than that observed on cells transfected with a vpu+ provirus. This increased expression of gp120 at the cell surface may explain the more rapid onset of syncytium formation observed in cell transfected with vpu mutant proviruses. These results suggest that Vpu-facilitated virion release and delayed cytopathic effect are the consequence of two distinct functional activities of the protein.
Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Québec, Canada.
J
J
Friborg
A
A
Ladha
H
H
Göttlinger
W A
WA
Haseltine
E A
EA
Cohen
D000595:Amino Acid Sequence; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002459:Cell Fusion; D002460:Cell Line; D016384:Consensus Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D015726:Giant Cells / Q000821:virology; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis; D015497:HIV-1 / Q000502:physiology*; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011533:Proviruses; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology; D000086082:Viroporin Proteins
D017931:DNA Primers; D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; D054298:Human Immunodeficiency Virus Proteins; D010446:Peptide Fragments; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins