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Title: Regulated Degradation of the HIV-1 Vpu Protein through a βTrCP-Independent Pathway Limits the Release of Viral Particles}
Article:
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.
Introduction:
The proteasome pathway ensures the degradation of proteins that are marked by poly-ubiquitination. This process is regulated by the E3 ubiquitin ligases that ensure the rate-limiting step of substrate selection[1]. So far, three classes of E3 ubiquitin ligases have been defined, based on the presence of a HECT, a Ring-H2-finger, and U-box domain, respectively. Within the Ring-H2-finger family are the Skp1-Cullin1-F-box proteins and the anaphase promoting complex/cyclosome complexes, which are both involved in cell-cycle progression[2]. The Skp1-Cullin1-F-box proteins display a modular organization consisting of constant core subunits( Skp1, Cullin1, Rbx1/Roc1) associated with a variable component, the F-box protein[3, 4]. The F-box protein binds Skp1 through its F-box motif and recruits the substrate through a second domain. More than 70 F-box proteins have been identified in humans, each member being responsible for the degradation of a specific subset of proteins[5]. In most cases, the binding of the F-box protein requires specific phosphorylation of the substrate[6]. For example, βTrCP requires phosphorylation of the serine residues present in the consensus motif DSGΦXS, which is found in most βTrCP substrates. Phosphorylation of the βTrCP recognition motif itself is often controlled by prior phosphorylation events of the substrate protein, which allows a very tight control of the degradation process[6, 7]. Viruses have developed multiple ways to subvert cell signaling pathways to replicate, disseminate, and escape from the host immune system. The diversion of the ubiquitin-mediated degradation machinery represents one such strategy, which has been first documented for the human papillomavirus E6 protein[8]. Our earlier study of the human HIV-1 viral protein U( Vpu) provided the first example reported for HIV-1[9], and there is now a growing list of proteins from various viruses that are reported to induce the degradation of cellular targets[10, 11]. Vpu is a small integral membrane protein encoded by HIV-1 and a subset of related simian immunodeficiency viruses and has no homolog in the less pathogenic HIV-2 virus[12, 13]. Recent studies in macaques using simian-HIV strains have shown that Vpu plays an active role in the pathogenesis[14]. Vpu performs two major functions in HIV-1 replication[15]. First, Vpu enhances the release of retroviral particles from most human cells, including lymphocytes and primary macrophages[16–22]. This activity of Vpu, which is not observed in simian cells, is suspected to reflect its ability to counteract a host cell restriction factor specific for human cells[23–25] and may depend on Vpu binding to the host channel TASK-1[26]. Second, Vpu induces the degradation of the CD4 viral receptor and therefore participates in the general downregulation of CD4 expression during the course of HIV infection[27]. Vpu-mediated CD4 degradation is thought to prevent CD4-Env binding in the endoplasmic reticulum in order to facilitate proper Env assembly into virions[28, 29]. We earlier reported the cloning and the characterization of βTrCP as a Vpu cell partner that the viral protein connects to CD4 to induce its degradation[9]. Interaction between βTrCP and Vpu, which controls the subsequent CD4 degradation, depends on the phosphorylation of serines 52 and 56 of Vpu within its DSGΦXS βTrCP recognition motif[9, 30]. In addition, the sequestration of βTrCP by Vpu has been shown to induce the stabilization of the βTrCP substrates, β-catenin and IκBα[31, 32]. We and others have previously observed that Vpu remains stable during the course of CD4 degradation[9, 33]. Here we report the unexpected finding that Vpu is subjected to proteasome-mediated degradation in cells arrested in early mitosis by nocodazole. We further show that this degradation process requires phosphorylation of the serine 61 residue adjacent to the βTrCP-binding motif. Therefore, differential phosphorylation of Vpu determines its fate as an adaptor or as a substrate of distinct ubiquitin ligases. The controlled degradation of Vpu, which is not mediated by βTrCP, decreases the efficiency of Vpu-mediated release of HIV-1 virions. It may thus contribute to the balance between efficient HIV-1 dissemination and preservation of the reservoir of infected cells.
Discussion:
Our initial goal was to study the potentially adverse effects of Vpu expression on the timely regulated degradation of βTrCP substrates involved in cell-cycle progression. To our surprise, Emi1 escaped the Vpu-mediated stabilization observed for the constitutively degraded βTrCP substrate, β-catenin, and for the cell-cycle regulator Cdc25A, normally degraded during the S phase. This paradox may be reconciled by our further demonstration of a regulated degradation process of Vpu itself, which predominantly occurs in early mitosis. Indeed, one simple explanation is that the pool of Vpu drops before the cell cycle reaches the phase where Emi1 degradation takes place, namely at the G2-M transition. Another nonexclusive possibility is the existence of a βTrCP pool dedicated to Emi1 degradation[39], which is nonaccessible to Vpu. We have shown that the phosphorylation of Vpu serine 61, which lies adjacent to the βTrCP recognition motif, is the triggering event for subsequent Vpu degradation. Accordingly, mutation of Vpu serine 61 into a non-phosphorylable residue stabilized Vpu, expressed either alone or in the context of the HIV-1. Two lines of evidence indicate that Vpu degradation occurs via a βTrCP-independent pathway:( i) The Vpu mutant, which fails to recruit βTrCP due to substitutions in its binding motif, was degraded in mitosis-arrested cells to the same extent as the wild-type protein;( ii) conversely, the S61 Vpu mutant, which still binds βTrCP, showed a much slower turnover than the wild-type protein. In turn, this implies that Vpu is recognized as a substrate by an E3 ubiquitin ligase other than βTrCP, as illustrated in the model presented in Figure 6. Vpu has been shown to be constitutively phosphorylated by casein kinase II on serine 52 and serine 56 of the βTrCP-binding motif[40]. As a result, Vpu binds βTrCP and acts as an adaptor between the SCFβTrCP and CD4, leading to the degradation of the HIV-1 receptor[9]. In contrast, the new phosphorylation event underscored here, which takes place on the serine 61, creates a binding site for an E3 ubiquitin ligase, which in turn targets Vpu for proteasome-mediated degradation. Phosphorylation of the serine 61 residue does not depend on prior phosphorylation of the βTrCP-binding motif since it still occurs when residues 52 and 56 are mutated. However, binding to the two E3 ubiquitin ligases might be mutually exclusive, considering the proximity between their respective binding sites. How the viral protein escapes the degradation via the SCFβTrCP ubiquitin ligase while it is recognized as a substrate by a second ubiquitin ligase remains an open question. It should be emphasized that such an issue holds for other viral proteins that subvert E3 ubiquitin ligases. For example, HPV E6 and E7 proteins show the same dual fate as Vpu: they act as adaptors of an E3 ubiquitin ligase to direct the abnormal degradation of a cellular protein for the benefit of the virus[11] and as substrates of a probably distinct E3 ubiquitin ligase[41–44]. In the same line, the HIV-1 Vpr protein, which recruits the DDB1-Cul4A E3 ubiquitin ligase to induce cell-cycle arrest as shown in our recent study[45], appears to be stabilized when incorporated in this complex( unpublished data). We show here that the slower turnover of Vpu resulting from S61 substitution increases the release of HIV-1 virions, hence suggesting a selective advantage. This prompted us to examine the degree of conservation of the corresponding Vpu residue among HIV-1 isolates. To address this point, we took advantage of the previous analysis performed by McCormick-Davis and coworkers( 2000) on the Vpu sequences from 100 strains of HIV-1 group M from diverse geographical regions[46]. Remarkably, 77% of these strains showed a serine residue, either at position 61 or at position 64, i. e., 4 and 7 residues downstream of the βTrCP-binding motif( Figure 6B and unpublished data). Of note, this spacing difference corresponds to one helix turn, which suggests that the relevant serine residue is presented on the same face of the Vpu α-helix. This indicates that within the M group of HIV-1, the evolutionary trend is toward the conservation of a regulated Vpu degradation process. HIV-1 replicates in both quiescent and dividing cells. In the latter, it might be advantageous for the virus to fine-tune virion production according to cell-cycle progression. This may in turn reflect that long-term persistence of the virus requires the optimal balance between efficient viral production and host cell viability. In light of our present study, the regulated degradation process of Vpu is expected to be lost in the viral strains naturally mutated at the relevant phosphorylation site. Whether these strains show higher pathogenicity will be interesting to address for our better understanding of the constraints that govern the relation between HIV-1 and the human organism.
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 .