Human Immunodeficiency Virus Type 1 Gag Engages the Bro1 Domain of ALIX/AIP1 through the Nucleocapsid

Abstract

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses harbor short peptide motifs in Gag that promote the release of infectious virions. These motifs, known as late assembly (L) domains, recruit a cellular budding machinery that is required for the formation of multivesicular bodies (MVBs). The primary L domain of HIV-1 maps to a PTAP motif in the p6 region of Gag and engages the MVB pathway by binding to Tsg101. Additionally, HIV-1 p6 harbors an auxiliary L domain that binds to the V domain of ALIX, another component of the MVB pathway. We now show that ALIX also binds to the nucleocapsid (NC) domain of HIV-1 Gag and that ALIX and its isolated Bro1 domain can be specifically packaged into viral particles via NC. The interaction with ALIX depended on the zinc fingers of NC, which mediate the specific packaging of genomic viral RNA, but was not disrupted by nuclease treatment. We also observed that HIV-1 zinc finger mutants were defective for particle production and exhibited a similar defect in Gag processing as a PTAP deletion mutant. The effects of the zinc finger and PTAP mutations were not additive, suggesting a functional relationship between NC and p6. However, in contrast to the PTAP deletion mutant, the double mutants could not be rescued by overexpressing ALIX, further supporting the notion that NC plays a role in virus release.Retroviral Gag polyproteins harbor short peptide motifs that promote the detachment of assembled virions from the cell surface and from each other (16,22,24,41,49,50,52). These motifs, which are known as late assembly (L) domains, often remain functional even if moved to a different position within Gag and can be exchanged among unrelated viruses (39, 51). In the case of human immunodeficiency virus type 1 (HIV-1), the primary L domain consists of a conserved P(T/S)AP motif near the beginning of the C-terminal p6 domain of the Gag polyprotein (22,24). The PTAP motif serves as a binding site for the host protein Tsg101, which is required for the release of infectious virions (21,31,47).Tsg101 is a component of the endosomal sorting complex ESCRT-I, which functions in the sorting of ubiquitinated cargo into small vesicles that bud into the lumen of multivesicular bodies (MVBs) (3,23,27). This invagination event is topologically related to retrovirus budding and ultimately serves to deliver transmembrane proteins to lysosomal compartments for degradation. The components of this cellular budding pathway were initially identified in Saccharomyces cerevisiae, where a protein network that includes at least 18 different vacuolar protein-sorting (Vps) proteins is required for the formation of MVBs (3). The absence of any of these gene products causes the formation of an abnormal endosomal structure called the class E compartment (26). The MVB sorting pathway is conserved throughout eukaryotic evolution, and at least one homolog for each of the yeast class E Vps proteins exists in humans (48). Most of the class E Vps proteins participate in the formation of ESCRT-I or of two o...

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

“…2E and column 5 of Fig. 2G), indicating that the reported interaction between the NC domain of Gag and Bro1 domain of ALIX (45,46) was not sufficient to recruit detectable levels of ALIX in this system. The ALIX PRD contains a P(S/T)AP motif that is capable of binding to the TSG101 subunit of ESCRT-I (18), although the motif is not essential for ALIX function in HIV-1 budding (19,20).…”

ALIX/AIP1 Is Required for NP Incorporation into Mopeia Virus Z-Induced Virus-Like Particles

“…2A). The YLCL motif could potentially interact with AIP1, which was also demonstrated to bind viral nucleocapsid proteins (31,37) and to regulate the transport of nucleocapsids from endosomes to the cytosol (25). To test whether this YLCL motif plays a role in VLP release or NP incorporation into VLPs, we generated a Z mutant in which YLCL was changed to AAAA ( Fig.…”

“…Analysis of Marburg virus NP revealed several putative late domains, one of which recruits TSG101 to the budding sites, resulting in enhanced VLP formation (12). Similarly, the zinc finger of the HIV-1 nucleocapsid domain of Gag has been shown to bind AIP1, cooperating with the late domains to employ the ESCRT pathway for viral budding (13,37). Here, we demonstrated that Mopeia virus uses an ESCRT-associated protein, AIP1, for NP incorporation into virions; the use of the ESCRT pathway for NP incorporation into virions has not been shown for any other virus.…”

“…ESCRT-II then activates ESCRT-III, which is located on endosomal membranes and is an essential part of the apparatus that induces MVB vesicle formation (1,2). For several viruses, AIP1 is known to bind to the TSG101 of ESCRT-I and to components of ESCRT-III, joining the two complexes and eliminating the need for ESCRT-II (13,28,37). AIP1 also recruits NEDD4-like proteins to facilitate HIV-1 release (42).…”

Socs1: A Host Factor Required for HIV-1 Gag Trafficking

“…Evidence suggests that Gag ubiquitination is important for virion assembly and viral infectivity [11,10]. Ubiquitination is thought to allow Gag to interact with and co-opt members of the endosomal sorting system (the ESCRT complexes [14]) for the purposes of virion production [9,7,21]. The trans-Golgi network-associated protein SH3RF1 (hPOSH) is the only E3 ubiquitin ligase known to directly interact with Gag, but it's unlikely to ubiquitinate Gag itself [1].…”