title: Vpu-mediated CD4 down-regulation and degradation is conserved among highly divergent SIV(cpz) strains.
abstract: Human immunodeficiency virus type 1 (HIV-1) along with simian immunodeficiency viruses from chimpanzees (SIV(cpz)) and three species of Old World monkeys from the genus Cercopithecus have been shown to encode a Vpu protein. To date, the functional characterization of Vpu has been limited to a small number of subtype B and more recently subtype C Vpu proteins. Using a recently developed VpuEGFP reporter system, we have shown that the subtype B and C Vpus are capable of preventing CD4 from being expressed on the cell surface. Using the same reporter system, we report here on the expression and functional analysis of Vpu protein from four SIV(cpz) isolates (CAM13, ANT, TAN1, and GAB1). All four SIV Vpu fusion proteins were efficiently expressed and prevented CD4 expression on the cell surface and induced CD4 degradation. This was surprising as three of the SIV(cpz) Vpu fusion proteins had only one canonical casein kinase II (CK-II) site (CAM13, ANT, TAN1) while previous studies with laboratory adapted HXB2 had indicated that both CK-II sites were required for CD4 degradation. Both ANT and TAN1 Vpu sequences encoded five consecutive negatively charged amino acids residues following the only CKII site (SAIEEDEE for ANT; SGVEEDEE for TAN1). We thus explored the possibility that this stretch of negatively charged amino acids might substitute for the lack of second CK-II site. Substitution of the aspartic acid at position 61 and glutamic acid at position 63 in the SIV(cpz) ANT Vpu within with lysine residues abolished the ability of this protein to down-modulate cell surface expression of CD4. Similarly, change of a serine to an alanine residue following the single consensus CK-II site of the CAM13 Vpu (SGNESDGGEEE) abolished CD4-down-regulation, suggesting that this serine was phosphorylated in the absence of a canonical CK-II site. Our results indicate that the serine was required, suggesting that this serine was phosphorylated by CK-II or possibly another cellular kinase. Taken together, these results show for the first time that Vpu proteins from SIV(cpz) isolates, although quite diverse in sequence and predicted secondary structure from the HIV-1 subtype B protein, are capable of down-regulating CD4, which is one of the major functions of the HIV-1 protein.
authors.affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Gomez
authors.forename: Erik
authors.initials: E
authors.lastname: Pacyniak
authors.forename: Melissa
authors.initials: M
authors.lastname: Flick
authors.forename: David R
authors.initials: DR
authors.lastname: Hout
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Gomez
authors.forename: Eric
authors.initials: E
authors.lastname: Nerrienet
authors.forename: Ahidjo
authors.initials: A
authors.lastname: Ayouba
authors.forename: Mario L
authors.initials: ML
authors.lastname: Santiago
authors.forename: Beatrice H
authors.initials: BH
authors.lastname: Hahn
authors.forename: Edward B
authors.initials: EB
authors.lastname: Stephens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D002679:Pan troglodytes / Q000821:virology; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015302:Simian Immunodeficiency Virus / Q000472:pathogenicity*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D015704:CD4 Antigens; 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