The Phe105 Loop of Alix Bro1 Domain Plays a Key Role in HIV-1 Release

Abstract

Summary Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects due to interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical “boomerang” folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently mutation of Phe105 and surrounding residues at the tip of the loop compromises the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.

Budding of Retroviruses Utilizing Divergent L Domains Requires Nucleocapsid

“…We previously presented evidence supporting a role for HIV-1 NC in the Alix-binding LYPX n L L domain budding pathway. We found that overexpression of the Alix Bro1 domain rescued virus release defects of an HIV-1 mutant lacking all L domains (26,67). Similar results were obtained with the SIVcpzGAB2 mutant lacking all L domains (data not shown).…”

“…Moreover, we and others have shown that NC interacts with the Bro1 domain of Alix (26,62), which mediates the recruitment of the cellular fission machinery components CHMP4 and VPS4 (26). Interestingly, this interaction is critical for the HIV-1 LYPX n L-driven budding pathway (26,62,66,67), emphasizing the role of NC in Alix-mediated HIV-1 budding. Several roles have been attributed to NC in the HIV-1 life cycle; they include reverse transcription, integration, trafficking, virus assembly, viral genome encapsidation, and cell-to-cell virus transmission (19,38,44,47,54,58,71).…”

“…However, a model involving a requirement of contacts between NC and the Bro1 domain of Alix during virus release is supported by the restoration of EIAV YPDL (lacking the YPDL motif) release following ectopic expression of the Bro1 domain and the latter's inability to rescue release when its binding to NC is compromised or eliminated (Fig. 5) (26,67), thus drawing a direct correlation between the efficiency of Bro1-NC interactions and Alix activity in virus release. Interestingly, Alix access to NC via intermolecular interaction(s) with coassembling Gag proteins-rather than intramolecular binding within the same Gag protein-appears to be sufficient for function.…”

“…The DNA of pGEX-NC-p9, RKI-p9, RKII-p9, and GST-empty-vector constructs was transformed into Escherichia coli BL21(DE3)/pLysS (Stratagene). GST pull-down assays were performed as previously described (67). Briefly, cultures in the exponential phase of growth were induced with isopropyl-␤-D-thiogalactopyranoside.…”

“…Viral release rescue events due to overexpression of Bro1 had been attributed to its ability to bind NC (26,67). To determine if NC is the actual domain required for Bro1 recruitment and rescue 1 and 2) or in the presence of increasing amounts of Nedd4.2s (lanes 3, 4, 6, and 7).…”