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IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULBP3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas.

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BackgroundHIV-1 relies on the host ESCRTs for release from cells. HIV-1 Gag engages ESCRTs by directly binding TSG101 or Alix. ESCRTs also sort ubiquitinated membrane proteins through endosomes to facilitate their lysosomal degradation. The ability of ESCRTs to recognize and process ubiquitinated proteins suggests that ESCRT-dependent viral release may also be controlled by ubiquitination. Although both Gag and ESCRTs undergo some level of ubiquitination, definitive demonstration that ubiquitin is required for viral release is lacking. Here we suppress ubiquitination at viral budding sites by fusing the catalytic domain of the Herpes Simplex UL36 deubiquitinating enzyme (DUb) onto TSG101, Alix, or Gag.ResultsExpressing DUb-TSG101 suppressed Alix-independent HIV-1 release and viral particles remained tethered to the cell surface. DUb-TSG101 had no effect on budding of MoMLV or EIAV, two retroviruses that rely on the ESCRT machinery for exit. Alix-dependent virus release such as EIAV’s, and HIV-1 lacking access to TSG101, was instead dramatically blocked by co-expressing DUb-Alix. Finally, Gag-DUb was unable to support virus release and dominantly interfered with release of wild type HIV-1. Fusion of UL36 did not effect interactions with Alix, TSG101, or Gag and all of the inhibitory effects of UL36 fusion were abolished when its catalytic activity was ablated. Accordingly, Alix, TSG101 and Gag fused to inactive UL36 functionally replaced their unfused counterparts. Interestingly, coexpression of the Nedd4-2s ubiquitin ligase suppressed the ability of DUb-TSG101 to inhibit HIV-1 release while also restoring detectable Gag ubiquitination at the membrane. Similarly, incorporation of Gag-Ub fusion proteins into virions lifted DUb-ESCRT inhibitory effect. In contrast, Nedd4-2s did not suppress the inhibition mediated by Gag-DUb despite restoring robust ubiquitination of TSG101/ESCRT-I at virus budding sites.ConclusionsThese studies demonstrate a necessary and natural role for ubiquitin in ESCRT-dependent viral release and indicate a critical role for ubiquitination of Gag rather than ubiquitination of ESCRTs themselves.

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.

Rhesus macaque TRIM5␣ (rhTRIM5␣) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5␣ restriction occurs via a two-step process. The first step is restriction factor binding, which is sufficient to inhibit infection. The second step, which is sensitive to proteasome inhibition, prevents the accumulation of reverse transcription products in the target cell. However, because of the pleotropic effects of proteasome inhibitors, the molecular mechanisms underlying the individual steps in the restriction process have remained poorly understood. In this study, we have fused the small catalytic domain of herpes simplex virus UL36 deubiquitinase (DUb) to the N-terminal RING domain of rhTRIM5␣, which results in a ubiquitination-resistant protein. Cell lines stably expressing this fusion protein inhibited HIV-1 infection to the same degree as a control fusion to a catalytically inactive DUb. However, reverse transcription products were substantially increased in the DUb-TRIM5␣ fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5␣. Similarly, expression of DUb-rhTRIM5␣ resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5␣ induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5␣-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5␣ failed to activate NF-B signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection. IMPORTANCEThese studies provide direct evidence that ubiquitin conjugation to rhTRIM5␣-containing complexes is required for the second step of HIV-1 restriction. They also provide a novel tool by which the biological activities of TRIM family proteins might be dissected to better understand their function and underlying mechanisms of action. TRIM5␣ is a retroviral restriction factor that mediates a postentry block to infection (1, 2). The best-studied example of this restriction is the ability of the TRIM5␣ protein from rhesus macaques (rhTRIM5␣) to potently inhibit HIV-1 infection (1, 2). As a member of the TRIM family of proteins, TRIM5␣ possesses the canonical RING, B-Box, and coiled-coil (CC) domains that comprise the tripartite motif (TRIM) that define this family of proteins (3). The N-terminal RING domain acts as an E3 ubiquitin ligase (4-6), and together with the B-Box domain, also functions to mediate the self-association of TRIM5␣ dimers (7-9). The CC domain, in cooperation with the Linker2 (L2) region, mediates the dimerization of TRIM5␣ monomers and the formation of higher-order assemblies (10-14). TRIM5␣ also possesses a C-terminal SPRY domain which is known to recognize determinants in the assembled viral core to mediate restriction (15-17). Following core binding, TRIM5␣ induces the abortive disassembly of the viral core (18,19). The mechanism by which core disruption o...

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.

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...