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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.
Journal of Virology 08/2012; 86(21):11608-15. · 5.40 Impact Factor
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ABSTRACT: Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic α helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an α helix, the CHMP5 C-terminal tail adopts a tandem β-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a β-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.
Structure 04/2012; 20(5):887-98. · 6.35 Impact Factor
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ABSTRACT: We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX(n)L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX(n)L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.
Journal of Virology 02/2012; 86(8):4182-93. · 5.40 Impact Factor
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ABSTRACT: 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 that result from 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 compromise 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.
Structure 09/2011; 19(10):1485-95. · 6.35 Impact Factor
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ABSTRACT: In addition to PTAP L domains, primate lentiviruses carry Alix-binding motifs that include the recently described type 3 SREKPYKEVTEDLLHLNSLF sequence. We examined the requirements for the type 3 sequence motif in simian immunodeficiency virus SIV(smE543) and identified the (499)LNSLF(503) sequence as a key functional determinant. Mutation of distal leucines (499)L and (502)L (LL mutant) caused an inhibitory effect on Alix-dependent SIV(smE543) release that was quantitatively similar to that observed following disruption of the type 3 L domain or RNA interference (RNAi) depletion of Alix. Similar results were obtained with the SIV(mac239) LL mutant. Thus, distal leucines are key determinants of SIV(smE543) and SIV(mac239) type 3 L domains.
Journal of Virology 08/2011; 85(21):11532-7. · 5.40 Impact Factor
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ABSTRACT: The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPXnL, that bind the cellular proteins Tsg101 and Alix, respectively. These interactions are thought to recruit members of the host fission machinery (ESCRT) to facilitate HIV-1 release. Here we report a new role for the p6-adjacent nucleocapsid (NC) domain in HIV-1 release. The mutation of basic residues in NC caused a pronounced decrease in virus release from 293T cells, although NC mutant Gag proteins retained the ability to interact with cellular membranes and RNAs. Remarkably, electron microscopy analyses of these mutants revealed arrested budding particles at the plasma membrane, analogous to those seen following the disruption of the PTAP motif. This result indicated that the basic residues in NC are important for virus budding. When analyzed in physiologically more relevant T-cell lines (Jurkat and CEM), NC mutant viruses remained tethered to the plasma membrane or to each other by a membranous stalk, suggesting membrane fission impairment. Remarkably, NC mutant release defects were alleviated by the coexpression of a Gag protein carrying a wild-type (WT) NC domain but devoid of all L domain motifs and by providing alternative access to the ESCRT pathway, through the in trans expression of the ubiquitin ligase Nedd4.2s. Since NC mutant Gag proteins retained the interaction with Tsg101, we concluded that NC mutant budding arrests might have resulted from the inability of Gag to recruit or utilize members of the host ESCRT machinery that act downstream of Tsg101. Together, these data support a model in which NC plays a critical role in HIV-1 budding.
Journal of Virology 03/2011; 85(5):2304-15. · 5.40 Impact Factor
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ABSTRACT: The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPX(n)L, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host's fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP(-)), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP(-) budding defects is independent of cellular Tsg101, implying that Nedd4-1's function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPX(n)L motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP(-). This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPX(n)L motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix's facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPX(n)L/Alix budding pathway via a mechanism that involves Alix ubiquitination.
Journal of Virology 08/2010; 84(16):8181-92. · 5.40 Impact Factor
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ABSTRACT: HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX(n)L, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Bro(i)) are sufficient to bind Gag. Bro(i) interferes with HIV-1 release in an NC-dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Bro(i) and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPX(n)L/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1-CHMP4 complex required for LYPX(n)L-mediated budding.
PLoS Pathogens 04/2009; 5(3):e1000339. · 9.13 Impact Factor
