Abstract
The cellular protein BST-2/CD317/Tetherin has been shown to inhibit the release of HIV-1 and other enveloped viruses from infected cells. The HIV-1 accessory protein Vpu binds to both BST-2 and βTrCP, a substrate-recognition subunit for the SCF (Skip1-Cullin1-F-box protein) E3 ubiquitin ligase complex. This interaction leads to both the degradation of BST-2 and the enhancement of viral egress. Recently BST-2 was shown to be ubiquitinated in this process. Here we have confirmed the Vpu- and βTrCP-dependent multi/polyubiquitination of BST-2. Ubiquitinated BST-2 accumulated in cells treated with a lysosomal inhibitor but not a proteasomal inhibitor. Additionally, we observed that a BST-2 mutant deleted for its cytosolically exposed lysine residues is also ubiquitinated. Subsequent experiments suggested that Vpu promotes BST-2 ubiquitination upon amino acid residues bearing hydroxyl- but not thiol-bearing side chains. However, a BST-2 mutant bearing substitutions for its cytoplasmically exposed Ser, Thr, and Lys residues was still down-regulated, ubiquitinated, and degraded in a Vpu-dependent manner. Our results suggest that Vpu may target either the BST-2 cytoplasmic Tyr residues or the NH(2) terminus itself for ubiquitination.
Highlights
HIV-1 Vpu enhances viral release by down-regulating the host restriction factor BST-2/Tetherin
These same high molecular weight (Mr) species were significantly enriched in cells expressing WT Vpu, indicating that BST-2 is ubiquitinated in the presence of Vpu
The main population of ubiquitinated products was centered around the 98-kDa molecular mass marker, and we cannot determine whether these ubiquitinated products result from the ϳ20- or ϳ35-kDa BST-2 forms, given a 10-kDa Ub monomer, we predict that these ubiquitinated forms have been modified with between 6 and 15 Ub monomers and are either multiply monoubiquitinated or polyubiquitinated
Summary
HIV-1 Vpu enhances viral release by down-regulating the host restriction factor BST-2/Tetherin. The HIV-1 accessory protein Vpu binds to both BST-2 and TrCP, a substrate-recognition subunit for the SCF (Skip1-Cullin1-F-box protein) E3 ubiquitin ligase complex This interaction leads to both the degradation of BST-2 and the enhancement of viral egress. The most well characterized target of the resulting Vpu-TrCP-SCF complex is the viral receptor CD4, which interacts with Vpu in the endoplasmic reticulum (ER) [7] This leads to the ubiquitination of CD4 [8, 9] on both lysine and serine/threonine residues located within its cytoplasmic tail. We find that Vpu can ubiquitinate and degrade a BST-2 mutant substituted for all lysines, serines, and threonines, suggesting that Vpu may instead target either tyrosines, cysteines, or the NH2-terminal methionine residue for ubiquitination
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