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To whom correspondence should be addressed: University of California, Mt. Zion Cancer Research Center, Rm. 226, Box 0703, 2340 Sutter St., San Francisco, CA 94143-0703. Tel.: 415-502-1902; Fax: 415-502-1901;
* This work was supported in part by grants from the European Molecular Biology Organization, the Peter und Traudl Engelhorn Stiftung (to M. G.), the Deutsche Forschungsgemeinschaft (to T. L. and O. T. F.), the University AIDS Research Program (to Y-H. Z.), the Howard Hughes Medical Institute, and National Institutes of Health Grant 1RO1AI38532-01.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors contributed equally to this work.
Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (μ2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and μ2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in μ2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.
human immunodeficiency virus type 1
armadillo repeat (helical structure segment of approximately 42 residues)
regulatory subunit H of the peripheral V1 domain of V-ATPases
simian immunodeficiency viruses
adaptor protein complex 2
fluorescence-activated cell sorter
Nef is a 27–35-kDa myristoylated, membrane-associated protein encoded by primate lentiviruses (HIV-1,1 HIV-2 and SIV). It is expressed abundantly early in the viral replicative cycle (
Targeting of proteins to endosomes largely depends on specific sorting signals. They include the tyrosine-based motif, YXXφ, where X and φ are any and bulky hydrophobic amino acids, respectively, and the dileucine-based motif, LL or Lφ, which often is preceded by an acidic residue at position −4 (
). The downstream motif, which is highly conserved among different nef alleles, is required for the internalization of CD4 by Nef as well as for its interaction with the subunit H of the vacuolar membrane ATPase, V1H (
). Thus, V1H could be a connector for Nef, which would carry CD4 from CCP to lysosomes for its degradation. To this end, we performed binding and internalization studies, which revealed that V1H binds Nef and the μ-chain of AP-2 in vitro and in vivo. In addition, when the C-terminal flexible loop in Nef was mutated or deleted, V1H fused to this truncated Nef protein could perform all the internalization functions of its wild-type counterpart. These findings fulfilled the structural and functional criteria for V1H as an adaptor between Nef and AP-2. Additionally, our results suggest that V1H plays a central role in the enhancement of virion infectivity by Nef.
In this study, we demonstrated that the regulatory subunit H of the V-ATPase binds the C-terminal flexible loop in Nef and the medium chain (μ2) of the adaptor protein complex AP-2. Thus, V1H can connect Nef and CD4 to the endocytic machinery. Direct and specific interactions between Nef and V1H, as well as V1H, and the μ2 chain could be demonstrated in vitro and in vivo. Importantly, binding sites on Nef, which bind CD4 and V1H, were separable so that distinct surfaces on Nef lacking the flexible loop, when fused to V1H, could still internalize CD4. To this end, we performed kinetic internalization assays on the receptor and Nef or fusion proteins between Nef and V1H. Indeed, Nef and V1H were found to traffic similarly to each other, which depended on the flexible loop in Nef. Finally, blocking the expression of V1H and thus the function of the V-ATPase decreased the infectivity of HIV.
Importantly, Nef and V1H decreased steady-state levels and increased rates of internalization of CD8 equivalently (Figs. 1 and 2). In the presence of only the N-terminal 160 residues of Nef, the hybrid mutant Nef160-V1H protein was also able to target CD4 as efficiently as the wild-type Nef protein. Thus, V1H was able to perform the function of the flexible loop in Nef, and the remainder of Nef was sufficient to bind to CD4. This finding is in complete agreement with structural studies between Nef and CD4 (
). Moreover, as reflected in the steady-state levels of CD4 and CD8, no significant recycling was observed with Nef or V1H fusion proteins.
Binding studies performed with GST-V1H fusion proteins and in vitro translated μ2 demonstrated that a central region of four ARM repeats of V1H interacts with the N-terminal domain in μ2 (Fig.5). Both protein fragments were stable and expressed to similar levels, which indicated a domain-domain protein interaction. The ARM or HEAT repeat superfold is composed of tandemly arranged helical repeats that form an elongated shaped molecule (
). This fold is known already from other proteins that are involved in intracellular trafficking processes, such as Importin α and β or β-Catenin. Its structural feature allows for sequence motif recognition by its concave surface and simultaneous assembly into multisubunit complexes. As an example, the binding site on Importin β for the small GTP-binding protein Ran does not overlap with its binding sites for the FxFG nucleoporin repeats but may instead generate a conformational change in the molecule (
By interacting with the N-terminal domain of μ2-(1–145), its ability to bind tyrosine-based sorting motifs for cargo uptake was not blocked. This suggests that a fully functional adaptor protein complex was preserved. However, we cannot exclude at this point that V1H might also substitute for the β-chain of the adaptor protein complex by its binding to μ2, generating thereby a complex with specific trafficking features. Because V1H is supposed to bind both V1 and V0 sectors of the vacuolar ATPase (
), its interaction with the adaptor protein complexes could also be important for the assembly of two ATPase sectors. Indeed, by its interaction with AP-2, V1H could also connect the V-ATPase to clathrin (
). With its binding to the flexible loop of Nef, V1H could thus function as an adaptor protein to mediate trafficking of CD4 and Nef to endosomes and lysosomes and thereby circumvent the transfer of cargo from adaptor complexes to coatomers (
). A model that displays the assembly of AP-2 complexes, the V-ATPase and clathrin at the plasma membrane, and the Nef-mediated internalization of CD4 is shown in Fig.7. The display of the V-ATPase is based on the latest models by electron microscopy (
Our results argue for an important role of the interaction between Nef and V1H for the enhancement of virion infectivity. This scenario is similar to that for SIV, where binding of Nef to V1H also correlated with the increased infectivity of virus particles (
). Thus, albeit the binding of V1H to the flexible loop in Nef facilitated the internalization of CD4, it also affected virion infectivity independently of CD4. What could be the mechanism of this effect of V1H? Consistent with the previous observation that the increase of virion infectivity by Nef is imprinted on the particle in the producing cell, our recent findings suggest that Nef acts as a chaperone of virus production by recruiting the assembly of HIV into lipid rafts (
), the adaptor function of V1H could facilitate the proper trafficking of a complex between Nef and viral structural proteins to the plasma membrane and their partitioning into lipid rafts, where local rearrangements of the actin cytoskeleton might facilitate particle release (
). As for CD4 down-regulation, it is unclear whether this would occur with or without involvement of the entire V-ATPase and its catalytic activity. Although it might be plausible that the recruitment of the V-ATPase could help to optimize the local pH that is required for the maturation of virus particles, no effect of Nef on maturation per se has been observed (
). Alternatively, Nef and V1H might trigger the internalization of yet unidentified cell surface receptors that counteract virion infectivity by mechanisms similar to CD4 down-regulation. These strategies might represent a variation on a common theme employed by other viruses such as HTLV-I and papillomaviruses that also engage the V-ATPase (
). Unraveling further details of the underlying mechanism will not only help us to understand viral pathogenesis, but also yield important insights into the role of the V-ATPase and its individual subunits in intracellular trafficking processes.
We thank John Guatelli and Juan Bonifacino for plasmids and Judith Gasteier for help with in vitro translation.