Abstract
The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein βTrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the βTrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both βTrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/βTrCP ubiquitin ligase.
Highlights
Human immunodeficiency virus-1 (HIV-1) Vpu is a small 81–86 amino acid accessory protein that enhances viral replication through the antagonism of a number of host cell proteins during infection [1,2]
We verified the role of the SCF/βTrCP1/2 E3 ubiquitin ligase as a critical cofactor for Vpu to block incorporation of mature, functional GaLV Env into pseudotyped HIV-1 particles
We further confirmed that this ligase is critical for Vpu to downmodulate CD4 from the cell surface and to antagonize the host restriction factor BST-2/tetherin
Summary
Human immunodeficiency virus-1 (HIV-1) Vpu is a small 81–86 amino acid accessory protein that enhances viral replication through the antagonism of a number of host cell proteins during infection [1,2]. Vpu has two key features that dictate functionality: an N-terminal transmembrane anchor that is capable of interacting with other transmembrane proteins, and a C-terminal cytoplasmic tail which contains two critical serine residues (S52,56 ) that are phosphorylated and can dictate interaction with various cellular proteins. The best studied targets of Vpu are CD4, which is the receptor for HIV-1, and BST-2/tetherin, an interferon-induced restriction factor which can prevent viral egress [3,4,5]. A cellular Skp, Cullin, F-Box (SCF) E3 ubiquitin ligase complex is recruited by Vpu to target CD4 Studies demonstrated that the membrane anchor sequence of Vpu could be scrambled without compromising the ability of Vpu to antagonize CD4, but changes to the cytoplasmic tail, S52,56 , abolished activity [4,6,7,8,9].
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