Regulated Degradation of the HIV-1 Vpu Protein through a βTrCP-Independent Pathway Limits the Release of Viral Particles

Abstract

Viral protein U (Vpu) of HIV-1 has two known functions in replication of the virus: degradation of its cellular receptor CD4 and enhancement of viral particle release. Vpu binds CD4 and simultaneously recruits the βTrCP subunit of the SCFβTrCP ubiquitin ligase complex through its constitutively phosphorylated DS52GXXS56 motif. In this process, Vpu was found to escape degradation, while inhibiting the degradation of βTrCP natural targets such as β-catenin and IκBα. We further addressed this Vpu inhibitory function with respect to the degradation of Emi1 and Cdc25A, two βTrCP substrates involved in cell-cycle progression. In the course of these experiments, we underscored the importance of a novel phosphorylation site in Vpu. We show that, especially in cells arrested in early mitosis, Vpu undergoes phosphorylation of the serine 61 residue, which lies adjacent to the βTrCP-binding motif. This phosphorylation event triggers Vpu degradation by a βTrCP-independent process. Mutation of Vpu S61 in the HIV-1 provirus extends the half-life of the protein and significantly increases the release of HIV-1 particles from HeLa cells. However, the S61 determinant of regulated Vpu turnover is highly conserved within HIV-1 isolates. Altogether, our results highlight a mechanism where differential phosphorylation of Vpu determines its fate as an adaptor or as a substrate of distinct ubiquitin ligases. Conservation of the Vpu degradation determinant, despite its negative effect on virion release, argues for a role in overall HIV-1 fitness.

Ubiquitination of BST-2 Protein by HIV-1 Vpu Protein Does Not Require Lysine, Serine, or Threonine Residues within the BST-2 Cytoplasmic Domain

“…This would make Vpu itself a likely target for SCF-dependent ubiquitination and turnover. However, it has also been reported that WT Vpu is as stable as Vpu2/6 (16), that Vpu is a stable, competitive inhibitor of ␤TrCP (13), that Vpu is degraded in a ␤TrCP-independent manner (14), that Vpu is degraded by the lysosome (15), and that Vpu is both polyubiquitinated in a ␤TrCP-dependent mechanism and degraded by the proteasome (12). Curiously, among the reports of Vpu stability, they also show direct evidence that Vpu2/6 is more stable that WT Vpu (70), suggesting that Vpu is in fact being turned over via a ␤TrCP-dependent mechanism.…”

“…CD4 is subsequently degraded by the proteasome (10) via what appears to be a noncanonical ER-associated degradation (ERAD) pathway (11). The fate of Vpu in this process is the subject of some debate, as it has been reported to either be exceptionally stable or to be ubiquitinated and degraded (12)(13)(14)(15)(16).…”

Role of retroviral restriction factors in the interferon-α–mediated suppression of HIV-1 in vivo

“…4A). Two of these mutations, A11G and S61A, occurred in highly conserved, previously established Vpu sequence domains involved in BST-2 down-regulation (39,40). We assessed the phenotypic consequence of these treatment-associated mutations by mutagenizing NL4-3 Vpu sequences at these positions and measuring the BST-2 down-regulation capacity of these mutant alleles in a subgenomic in vitro model (41).…”

Modulation of β-Catenin and E-Cadherin Interaction by Vpu Increases Human Immunodeficiency Virus Type 1 Particle Release

“…␤-TrCP binds to Vpu after casein kinase II-mediated phosphorylation of Vpu residues 52 and 56 (48). Vpu has been shown to stabilize several ␤-TrCP substrates, including the adherens junction protein ␤-catenin, NF-B inhibitor IB␣ (nuclear factor B), ATF4 (activating transcription factor 4), and cdc25A (cell division cycle protein 25A) (5,20). However, other ␤-TrCP substrates, such as Emi-1 (early mitotic inhibitor-1), remain unaffected by Vpu (20).…”

“…Vpu has been shown to stabilize several ␤-TrCP substrates, including the adherens junction protein ␤-catenin, NF-B inhibitor IB␣ (nuclear factor B), ATF4 (activating transcription factor 4), and cdc25A (cell division cycle protein 25A) (5,20). However, other ␤-TrCP substrates, such as Emi-1 (early mitotic inhibitor-1), remain unaffected by Vpu (20). Of note, Vpu binding to ␤-TrCP does not promote its own degradation, and in fact, Vpu is degraded through a ␤-TrCP-independent mechanism that depends on the phosphorylation of residue 61 (20).…”