HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observed that approximately 1–5% of CD4 + T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cell biology analyses, we discovered that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr formed a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhanced Plk4’s functionality by promoting its relocalization to the procentriole assembly and induced centrosome amplification. Loss of either Vpr’s C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogated Vpr’s capacity to induce all these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induced multiple centrosomes and aneuploidy in primary CD4 + T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.

Cyclophilin A (CypA) binds to the HIV-1 capsid to facilitate reverse transcription and nuclear entry and counter the antiviral activity of TRIM5 alpha. Interestingly, recent studies suggest that the capsid enters the nucleus of an infected cell and uncoats prior to integration. We have previously reported that the capsid protein regulates HIV-1 integration. Therefore, we probed whether CypA-capsid interaction also regulates this post-nuclear entry step. First, we challenged CypA-expressing (CypA(+/+)) and CypA-depleted (CypA(-/-)) cells with HIV-1 and quantified the levels of provirus. CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5 alpha. In addition, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited proviral integration in CypA(+/+) cells but not in CypA(-/-) cells. HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at the integration step in CypA(+/+) cells but not in CypA(-/-) cells. Then, to understand the mechanism, we assessed the integration activity of the HIV-1 preintegration complexes (PICs) extracted from acutely infected cells. PICs from CypA(-/-) cells retained lower integration activity in vitro compared to those from CypA(+/+) cells. PICs from cells depleted of both CypA and TRIM5 alpha also had lower activity, suggesting that CypA's effect on PIC was independent of TRIM5 alpha. Finally, CypA protein specifically stimulated PIC activity, as this effect was significantly blocked by CsA. Collectively, these results provide strong evidence that CypA directly promotes HIV-1 integration, a previously unknown role of this host factor in the nucleus of an infected cell.

Fluorosubstituted tryptophans serve as valuable probes for fluorescence and nuclear magnetic resonance (NMR) studies of proteins. Here, we describe an unusual photoreactivity introduced by replacing the single tryptophan in cyclophilin A with 7-fluoro-tryptophan. UV exposure at 282 nm defluorinates 7-fluoro-tryptophan and crosslinks it to a nearby phenylalanine, generating a bright fluorophore. The crosslink-containing fluorescent protein possesses a large quantum yield of ∼0.40 with a fluorescence lifetime of 2.38 ns. The chemical nature of the crosslink and the three-dimensional protein structure were determined by mass spectrometry and NMR spectroscopy. To the best of our knowledge, this is the first report of a Phe–Trp crosslink in a protein. Our finding may break new ground for developing novel fluorescence probes and for devising new strategies to exploit aromatic crosslinks in proteins.

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

High-resolution cryoET structures of native HIV-1 capsid in complex with IP6 and CypA are enabled by viral membrane perforation.

Background: Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods: HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results: We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether , our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps.

HIV-1 Vpr is a prototypic member of a large family of structurally related lentiviral virulence factors that antagonize various aspects of innate antiviral immunity. It subverts host cell DNA repair and protein degradation machineries by binding and inhibiting specific post-replication repair enzymes, linking them via the DCAF1 substrate adaptor to the Cullin 4 RING E3 ligase (CRL4 DCAF1 ). HIV-1 Vpr also binds to the multi-domain protein hHR23A, which interacts with the nucleotide excision repair protein XPC and shuttles ubiquitinated proteins to the proteasome. Here, we report the atomic resolution structure of Vpr in complex with the C-terminal half of hHR23A, containing the XPC-binding (XPCB) and ubiquitin-associated (UBA2) domains. The XPCB and UBA2 domains bind to different sides of Vpr’s 3-helix-bundle structure, with UBA2 interacting with the α2 and α3 helices of Vpr, while the XPCB domain contacts the opposite side of Vpr’s α3 helix. The structure as well as biochemical results reveal that hHR23A and DCAF1 use overlapping binding surfaces on Vpr, even though the two proteins exhibit entirely different three-dimensional structures. Our findings show that Vpr independently targets hHR23A- and DCAF1- dependent pathways and highlight HIV-1 Vpr as a versatile module that interferes with DNA repair and protein degradation pathways.

Oxidative chemical modifications of lipids and other biomolecules change their biophysical properties, but they are also increasingly recognized as crucial cellular quality control signals. The oxidation of mitochondrial lipids is an important trigger of apoptosis, in line with the observation that pathogenic mitochondrial dysfunction generates excessive reactive oxygen species (ROS). Remarkably, cells do not rely merely on incidental oxidative events, but actually facilitate the pro-active peroxidation of specific mitochondrial lipids. We use solid-state NMR to probe the molecular interactions that mediate the formation and activation of the protein-lipid complex behind this pro-apoptotic peroxidation reaction. We measure interactions between the protein (mitochondrial cytochrome c) and the lipid bilayer surface, interactions with solvent molecules, and the conformation and dynamics of the membrane-bound protein. We complement structural studies with assays of lipid-specific peroxidase activity under these sample conditions via mass spectrometry-based lipidomics, finding a recapitulation of the type of pro-apoptotic cardiolipin peroxidation seen in vivo. These functional properties display a notable dependence on the lipid types being bound and chemically modified by the ROS-mediated oxidation reactions. Via ssNMR we observe the structural and dynamic changes affecting the protein as it associates with different types of lipid bilayers. We previously reported that the activation of lipid peroxidase activity occurs with the native fold largely preserved. Spectral signatures of the membrane-bound protein are clearly distinct from those of denatured cytochrome c. Remarkably, localized spectral changes indicate selective dynamics in the protein-lipid complex that respond to the membrane fluidity. Different dynamic modulations occur dependent on the lipid composition, in terms of acyl chain and headgroup type. Thus, lipids act not only as preferred substrates but also as crucial dynamic regulators of this lethal interplay of proteins and lipids at the mitochondrial inner membrane.

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels,Escherichia coli(Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexamericEc-dGTPase, including an X-ray free-electron laser structure of the freeEc-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology,Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however,Ec-dGTPase residues at the end of the substrate-binding pocket mimic Watson–Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.

Comprehensive resonance assignments and delineation of the secondary structure elements of the C-terminal Vpr-binding region of hHR23A, residues 223–363, were achieved by triple-resonance NMR experiments on uniformly 13C,15N-labeled protein. Assignments are 100% and > 95% complete for backbone and side-chain resonances, respectively. This data constitutes important complementary information for our ongoing structure determination of the Vpr-hHR23A(223–363) complex. At high concentrations, severe line-broadening was observed for several residues in the 1H–15N HSQC spectrum, most likely resulting from inter-molecular interactions.

We report dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) NMR spectroscopy in viral capsids from HIV-1 and bacteriophage AP205. Viruses regulate their life cycles and infectivity through modulation of their structures and dynamics. While static structures of capsids from several viruses are now accessible with near-atomic-level resolution, atomic-level understanding of functionally important motions in assembled capsids is lacking. We observed up to 64-fold signal enhancements by DNP, which permitted in-depth analysis of these assemblies. For the HIV-1 CA assemblies, a remarkably high spectral resolution in the 3D and 2D heteronuclear data sets permitted the assignment of a significant fraction of backbone and side-chain resonances. Using an integrated DNP MAS NMR and molecular dynamics (MD) simulation approach, the conformational space sampled by the assembled capsid at cryogenic temperatures was mapped. Qualitatively, a remarkable agreement was observed for the experimental 13C/15N chemical shift distributions and those calculated from substructures along the MD trajectory. Residues that are mobile at physiological temperatures are frozen out in multiple conformers at cryogenic conditions, resulting in broad experimental and calculated chemical shift distributions. Overall, our results suggest that DNP MAS NMR measurements in combination with MD simulations facilitate a thorough understanding of the dynamic signatures of viral capsids.

The peroxidation of cardiolipins by reactive oxygen species, which is regulated and enhanced by cytochrome c (cyt c), is a critical signaling event in mitochondrial apoptosis. We probe the molecular underpinnings of this mitochondrial death signal through structural and functional studies of horse heart cyt c binding to mixed-lipid membranes containing cardiolipin with mono- and polyunsaturated acyl chains. Lipidomics reveal the selective oxidation of polyunsaturated fatty acid (PUFA) cardiolipin (CL), while multidimensional solid-state NMR probes the structure and dynamics of the membrane and the peripherally bound protein. The hydrophilic milieu at the membrane interface stabilizes a native-like fold, but also leads to localized flexibility at the membrane-interacting protein face. PUFA CL acts as both a preferred substrate and a dynamic regulator by affecting the dynamics of the cyt c N70-I85 Ω loop, which covers the heme cavity.

The interaction of cytochrome c (cytc) and cardiolipin (CL), a mitochondrial specific anionic lipid, has been identified as critical to initialize mitochondrial apoptosis. To fulfill this function, cytc and CL form a complex with significant CL peroxidase activity. Understanding the structural specificity of the cytc/CL complex is crucial to drug development targeting early apoptosis in cancer and cardiovascular diseases. In our studies, we constitute an in vitro functional assay designed to mimic cytc and CL in the proapoptotic stage and are able to quantify oxygenated molecular species of CL by lipidomics analysis using mass spectroscopy. Moreover, the structure and dynamics of peroxidase-active cytc bound to CL are investigated by both through-space and through-bond correlation experiments of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. Residues showing strong structural perturbations upon binding to CL are identified. Remarkably, cytc in complex with CL is associated with elaborate dynamics that indicate structural plasticity. A specific region of cytc is found to exhibit both significant structural and dynamics perturbations. In general, our results support a model of interaction consisting of a clustering of CL molecules while cytc retains much of its native fold and is bound to the membrane surface. The cytc dynamic changes and structural plasticity may have a strong implication for its proapoptotic activity.

AbstractDeoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels,Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly-located on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 Å) of single stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.Significance StatementdNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 –which blocks reverse transcription of HIV-1 in macrophages– Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP.

HIV-1 polymerase reverse transcribes the viral RNA genome into imperfectly double-stranded proviral DNA, containing gaps and flaps, for integration into the host cell chromosome. HIV-1 reverse transcripts share characteristics with cellular DNA replication intermediates and are thought to be converted into fully double-stranded DNA by cellular postreplication DNA repair enzymes. Therefore, the finding that the HIV-1 accessory protein Vpr antagonizes select postreplication DNA repair enzymes that can process HIV-1 reverse transcripts has been surprising. Here, we show that one such Vpr-antagonized enzyme, exonuclease 1, inhibits HIV-1 replication in T cells. We identify exonuclease 1 as a member of a new class of HIV-1 restriction factors in T cells and propose that certain modes of DNA “repair” inhibit HIV-1 infection.

The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid's hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid's morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.