The human Bro1 domain proteins include ALIX, HD-PTP, Brox, and the closely related RHP1 and RHP2 proteins. HIV-1 engages ALIX through its LYPxn
L-type L domain in p6, which serves as a docking site for the central V domain of ALIX (17
). Recently, it emerged that ALIX additionally interacts with the NC region of HIV-1 Gag through its N-terminal Bro1 domain (37
). We now report that Brox, a Bro1 domain-only protein, is also capable of interacting with HIV-1 NC, as are the isolated Bro1 domains of HD-PTP and of RPH1. While HD-PTP is a paralog of ALIX, RHP1 has a different domain organization. Also, the Bro1 domains of Brox and RHP1 exhibit only limited sequence homology to that of ALIX. Our results thus reveal that HIV-1 NC can interact with widely divergent Bro1 domains.
The ALIX-NC interaction depends on conserved zinc finger motifs in NC (37
), which are thought to mediate sequence-specific interactions with the packaging signal in the genomic viral RNA (2
). Nevertheless, nucleic acid does not appear to be required for the ALIX-NC interaction, because the in vitro interaction was insensitive to nuclease treatment (37
). The present study indicates that the zinc fingers in NC have a general affinity for Bro1 domains, since the disruption of both zinc fingers by the C28,49S
mutation interfered with the incorporation of Brox and of the isolated Bro1 domains of RHP1 and HD-PTP into VLP. Similarly, the C28,49S
mutation interferes with the binding of ALIX to NC (37
). Of note, in a WT HIV-1 context, the C28,49S
mutation caused a defect in viral particle production and a Gag processing defect that resembled that of HIV-1 L domain mutants (12
). On the other hand, the C28,49S
mutation did not further reduce particle production in the context of an HIV-1 L domain mutant, which suggested that the NC zinc fingers and the L domain are required for the same step in virus assembly (37
The membrane fission defect of HIV-1 mutants that lack the P(T/S)AP motif required for Tsg101 binding can be fully corrected by overexpressing ALIX (16
). In contrast, the isolated Bro1 domain of ALIX was inactive in the ΔPTAP rescue assay (37
), as expected since both the V domain and the C-terminal proline-rich domain of ALIX are required for its ability to rescue Tsg101 binding site mutants (6
). Similarly, in the present study, the overexpression of the Bro1 domain-only protein Brox did not significantly enhance VLP production by ΔPTAP HIV-1. However, we found that Brox is as potent as the isolated ALIX Bro1 domain in rescuing a minimal Gag construct that retains the PTAP L domain but lacks the binding site for the V domain of ALIX. Furthermore, the isolated Bro1 domains of HD-PTP and of RPH1 also exhibited activity in this assay. Apart from the ALIX binding site in p6, the minimal Gag construct used in these experiments lacked all Gag-Gag interaction sites in the N-terminal half of the Gag precursor and thus was presumably attenuated in its ability to bend the plasma membrane away from the cytosol. One possible interpretation of our results is that isolated Bro1 domains, although unable to trigger the membrane fission event required for viral particle release, can nevertheless assist Gag in the deformation of cellular membranes.
The observation that all Bro1 domains tested were active in the minimal Gag release assay pointed to the involvement of a feature that is shared among widely divergent Bro1 domains. One such feature appears to be an interaction site for CHMP4 family members (26
). For instance, the Bro1 domains of HD-PTP and of Brox both interact with CHMP4B (23
), and binding to CHMP4 has been shown to be essential for biological function in the case of HD-PTP (14
). Although RPH2 did not interact with human CHMP4 proteins in one study (35
), recent results obtained with ALIX raise the possibility that the docking site for CHMP4 can be occluded by autoinhibition (48
). Surprisingly, we found that point mutations in Brox that prevented its interaction with CHMP4B also reduced its NC-dependent uptake into HIV-1 particles. Since the steady-state levels of Brox were unaffected, this observation raises the possibility that the CHMP4 binding site contributed to the interaction with Gag. Interestingly, Brox mutants that showed no interaction with CHMP4B nevertheless retained considerable activity in the minimal Gag rescue assay, indicating that this activity may be intrinsic to the Bro1 domain itself. However, we cannot exclude that these Brox mutants retained some ability to bind CHMP4 in vivo, even though no binding was evident in our coprecipitation assay.
The Bro1 domain has the shape of a boomerang, which could theoretically generate negative curvature by interacting with the cytosolic face of cellular membranes through its convex surface (16
). Consistent with this possibility, the convex face of the Bro1 domain of a yeast homolog of ALIX possesses a highly basic patch that could interact with anionic phospholipids (26
), and this basic patch is to some extent conserved in ALIX (16
). Furthermore, ALIX binds to bilayers that contain the unconventional phospholipid lysobisphosphatidic acid, which is thought to regulate the formation of invaginations with negative curvature at the limiting membrane of late endosomes (15
An involvement of cellular proteins in the generation of membrane curvature by Gag is suggested by reports showing that the PPXY-type L domains of human T-cell leukemia virus type 1 and of Mason-Pfizer monkey virus are required early during bud formation (20
). More specifically, there is evidence that ALIX plays a role in membrane deformation by equine infectious anemia virus Gag, because budding was arrested at an early stage in the presence of dominant-negative ALIX (41
). In light of this observation, we propose that widely divergent Bro1 domains stimulate VLP production by a partially defective minimal Gag molecule because they share the capacity to interact with HIV-1 Gag and to generate negative membrane curvature.