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

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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. In addition to the structural and catalytic proteins characteristic of retroviruses, HIV-1 encodes auxiliary proteins that govern virus-host interaction. One such auxiliary protein termed Vpu both enhances virion release from human cells and the degradation of CD4, the cellular surface receptor of HIV-1. The latter activity relies on a strategy frequently used by pathogens, i.e., the hijacking of the protein degradation machinery. This process involves the ubiquitin ligases that ensure the selection of the protein subsequently degraded. Vpu interacts both with an ubiquitin ligase and CD4, bridging the latter to the degradation machinery. Here, we show that in addition to subverting one particular ubiquitin ligase to trigger CD4 degradation, Vpu is itself recognized in a phosphorylation-dependent manner by a distinct ubiquitin ligase and degraded. Mutations of Vpu that confer resistance to this degradation process enhance viral particle release, whereas the determinant of Vpu turnover is paradoxically well conserved among HIV-1 isolates. These results suggest that the virus is constrained to limit its own production for long-term persistence.