Identification of the HIV-1 NC Binding Interface in Alix Bro1 Reveals a Role for RNA

Abstract

HIV-1 recruits members of ESCRT, the cell membrane fission machinery that promotes virus exit. HIV-1 Gag protein gains access to ESCRT directly by binding Alix, an ESCRT-associated protein that promotes budding. The Alix Bro1 and V domains bind Gag NC and p6 regions, respectively. Whereas V-p6 binding and function are well characterized, residues in Bro1 that interact with NC and their functional contribution to Alix-mediated HIV-1 budding are unknown. We mapped Bro1 residues that constitute the NC binding interface and found that they are critical for function. Intriguingly, residues involved in interactions on both sides of the Bro1-NC interface are positively charged, suggesting the involvement of a negatively charged cellular factor serving as a bridge. Nuclease treatment eliminated Bro1-NC interactions, revealing the involvement of RNA. These findings establish a direct role for NC in mediating interactions with ESCRT necessary for virus release and report the first evidence of RNA involvement in such recruitments. HIV-1 usurps members of the host cell fission machinery to promote virus release. Two conserved sequences located within the C-terminal p6 domain of Gag, PTAP, and LYPXnL, named late (L) domains, are utilized to fulfill such functions. They bind Tsg101 and Alix, respectively (14,37,40), two host cellular proteins that initiate a set of sequential interactions leading to the recruitment of members of the endosomal sorting complex required for transport (ESCRT) pathway (5, 9, 30). The latter is comprised of three multiprotein complexes, named ESCRT-I, ESCRT-II, and ESCRT-III, that facilitate membrane-modeling events critical for multivesicular body (MVB) generation (2, 3), cytokinesis (7), and autophagy (32).Tsg101 functions in HIV-1 release as part of ESCRT-I (26) and mediates access to members of ESCRT-III, the charged MVB protein CHMP2 and CHMP4 isoforms, as well as the VPS4 ATPase (29,38,41). Whereas interactions that link Tsg101 (and ESCRT-I) to ESCRT-III are still unknown, Alix binds CHMP4 isoforms directly, thus linking Gag to 19,37,39). Although the Tsg101/PTAP pathway is considered predominant in HIV-1 release, the Alix/LYPXnL pathway is also functional in 293T cells and appears to be more efficient in T lymphocytes (11-13, 37, 39). This pathway is also sufficient to drive the release of the equine infectious anemia virus (EIAV) (8, 37), a lentivirus that relies solely on cellular Alix for virus budding.Alix structure revealed two well-ordered domains, the N-terminal boomerang-shaped Bro1 and the central V-shaped domains (12, 21); they interact with the NC and p6 domains of HIV-1 Gag, respectively (10-12, 31, 37). The binding interface between the LYPXnL motif and the V domain and its functional role have been well characterized (12). In contrast, residues in the Alix Bro1 domain that mediate interactions with NC (10, 11, 31) and their role in virus release are not known. We performed a mutational analysis and used a combination of binding and functional assays to map the Bro1-NC interface...

Advances in targeting nucleocapsid–nucleic acid interactions in HIV-1 therapy

“…When NC is contained within the precursor Gag polyprotein, the NC domain is involved in genomic RNA packaging, Gag-Gag multimerization and possibly initiation of particle production during virus assembly. The NC domain also plays a pivotal role in recruiting host cellular factors such as the members of the endosomal sorting complex required for transport (ESCRT) machinery that promote viral release (Sette et al, 2012). The mature NC protein, which is formed upon protease-mediated cleavage of the Gag polyprotein, facilitates encapsidation of the nucleic acids and is found in tight association with viral RNA in the viral core.…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…The first 202 positions in the Brol1 domain of AIP1 bind to the zinc finger and N-terminal domains of NC Gag (376,378,381), particularly key positions such as R3, R7, R10, K11, K14, K20, and R26 (382). Data from crystallization analyses also suggest that amino acid position F105 at the unique extended loop of AIP1 is crucial for HIV-1 budding (383).…”

“…Notably, the NC Gag -AIP1 interaction re-quires the involvement of RNA (376) and the cellular protein galectin-3 (391). In the former case, viral RNA bridges the interaction between NC Gag and AIP1 (376). In the latter case, galectin-3 interacts with AIP1 to promote the AIP1-p6…”

“…Although a direct interaction between Gag and Nef has not been reported to our knowledge, both NC Gag and Nef bind to AIP1 for efficient viral budding (375)(376)(377)(378). AIP1 (apoptosis-linked gene 2 [ALG2]-interacting protein 1) (also known as Alix or PDCD6IP) is associated with the endosomal sorting complex required for transport (ESCRT) machinery (377).…”

“…Regarding the interaction domains, the Bro1 and V domains of AIP1 can bind to NC Gag and p6 Gag , respectively (376,378). The N-terminal basic residues and zinc finger domains of NC Gag interact with AIP1 (381).…”