ABSTRACT
The dynamics and regulation of HIV-1 nuclear import and its intranuclear movements after import have not been studied. To elucidate these essential HIV-1 post-entry events, we labeled viral complexes with two fluorescently tagged virion-incorporated proteins (APOBEC3F or integrase), and analyzed the HIV-1 dynamics of nuclear envelope (NE) docking, nuclear import, and intranuclear movements in living cells. We observed that HIV-1 complexes exhibit unusually long NE residence times (1.5±1.6 hrs) compared to most cellular cargos, which are imported into the nuclei within milliseconds. Furthermore, nuclear import requires HIV-1 capsid (CA) and nuclear pore protein Nup358, and results in significant loss of CA, indicating that one of the viral core uncoating steps occurs during nuclear import. Our results showed that the CA-Cyclophilin A interaction regulates the dynamics of nuclear import by delaying the time of NE docking as well as transport through the nuclear pore, but blocking reverse transcription has no effect on the kinetics of nuclear import. We also visualized the translocation of viral complexes docked at the NE into the nucleus and analyzed their nuclear movements and determined that viral complexes exhibited a brief fast phase (<9 min), followed by a long slow phase lasting several hours. A comparison of the movement of viral complexes to those of proviral transcription sites supports the hypothesis that HIV-1 complexes quickly tether to chromatin at or near their sites of integration in both wild-type cells and cells in which LEDGF/p75 was deleted using CRISPR/cas9, indicating that the tethering interactions do not require LEDGF/p75. These studies provide novel insights into the dynamics of viral complex-NE association, regulation of nuclear import, viral core uncoating, and intranuclear movements that precede integration site selection.
Subject(s)
Cell Nucleus/metabolism , HIV Infections/metabolism , HIV-1/metabolism , Virus Integration/physiology , Virus Uncoating/physiology , Active Transport, Cell Nucleus/physiology , Blotting, Western , Cell Line , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Microscopy, Confocal , Nuclear Envelope/metabolism , Nuclear Pore Complex Proteins/metabolismABSTRACT
UNLABELLED: Rhesus macaque TRIM5α (rhTRIM5α) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5α 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 rhTRIM5α, 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-TRIM5α fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5α. Similarly, expression of DUb-rhTRIM5α resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5α induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5α-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5α failed to activate NF-κB signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection. IMPORTANCE: These studies provide direct evidence that ubiquitin conjugation to rhTRIM5α-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.
Subject(s)
HIV-1/immunology , HIV-1/physiology , Proteins/metabolism , Reverse Transcription , Ubiquitin/metabolism , Virus Assembly , Animals , Capsid/metabolism , Cell Line , Humans , Macaca mulatta , Ubiquitin-Protein LigasesABSTRACT
UNLABELLED: TRIM5α proteins are a potent barrier to the cross-species transmission of retroviruses. TRIM5α proteins exhibit an ability to self-associate at many levels, ultimately leading to the formation of protein assemblies with hexagonal symmetry in vitro and cytoplasmic assemblies when expressed in cells. However, the role of these assemblies in restriction, the determinants that mediate their formation, and the organization of TRIM5α molecules within these assemblies have remained unclear. Here we show that α-helical elements within the Linker2 region of rhesus macaque TRIM5α govern the ability to form cytoplasmic assemblies in cells and restrict HIV-1 infection. Mutations that reduce α-helix formation by the Linker2 region disrupt assembly and restriction. More importantly, mutations that enhance the α-helical content of the Linker2 region, relative to the wild-type protein, also exhibit an increased ability to form cytoplasmic assemblies and restrict HIV-1 infection. Molecular modeling of the TRIM5α dimer suggests a model in which α-helical elements within the Linker2 region dock to α-helices of the coiled-coil domain, likely establishing proper orientation and spacing of protein domains necessary for assembly and restriction. Collectively, these studies provide critical insight into the determinants governing TRIM5α assembly and restriction and demonstrate that the antiviral potency of TRIM5α proteins can be significantly increased without altering the affinity of SPRY/capsid binding. IMPORTANCE: Many members of the tripartite motif (TRIM) family of proteins act as restriction factors that directly inhibit viral infection and activate innate immune signaling pathways. Another common feature of TRIM proteins is the ability to form protein assemblies in the nucleus or the cytoplasm. However, the determinants in TRIM proteins required for assembly and the degree to which assembly affects TRIM protein function have been poorly understood. Here we show that alpha helices in the Linker2 (L2) region of rhesus TRIM5α govern assembly and restriction of HIV-1 infection. Helix-disrupting mutations disrupt the assembly and restriction of HIV-1, while helix-stabilizing mutations enhance assembly and restriction relative to the wild-type protein. Circular dichroism analysis suggests that that the formation of this helical structure is supported by intermolecular interactions with the coiled-coil (CC) domain in the CCL2 dimer. These studies reveal a novel mechanism by which the antiviral activity of TRIM5α proteins can be regulated and provide detailed insight into the assembly determinants of TRIM family proteins.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , HIV-1/genetics , HIV-1/metabolism , Protein Structure, Secondary/genetics , Animals , Cell Line , Cell Line, Tumor , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , HEK293 Cells , HeLa Cells , Humans , Macaca mulatta/genetics , Macaca mulatta/microbiology , Macaca mulatta/virology , Mutation/geneticsABSTRACT
BACKGROUND: The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues. RESULTS: The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions. CONCLUSIONS: Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction.
Subject(s)
Carrier Proteins/metabolism , HIV-1/metabolism , Proteasome Endopeptidase Complex/metabolism , ATPases Associated with Diverse Cellular Activities , Antiviral Restriction Factors , Carrier Proteins/genetics , Cytoplasm/genetics , Cytoplasm/metabolism , Cytoplasm/virology , DNA, Recombinant/genetics , DNA, Recombinant/metabolism , Fluorescence Resonance Energy Transfer , Gene Library , HEK293 Cells , HIV Infections/metabolism , HIV Infections/virology , HIV-1/pathogenicity , Humans , Immunoprecipitation , Microscopy, Fluorescence , Proteasome Endopeptidase Complex/genetics , Protein Binding , Protein Interaction Mapping/methods , Species Specificity , Transfection , Tripartite Motif Proteins , Two-Hybrid System Techniques , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases , UbiquitinationABSTRACT
The TRIM5α protein from rhesus macaques (rhTRIM5α) mediates a potent inhibition of HIV-1 infection via a mechanism that involves the abortive disassembly of the viral core. We have demonstrated that alpha-helical elements within the Linker 2 (L2) region, which lies between the SPRY domain and the Coiled-Coil domain, influence the potency of restriction. Here, we utilize single-molecule FRET analysis to reveal that the L2 region of the TRIM5α dimer undergoes dynamic conformational changes, which results in the displacement of L2 regions by 25 angstroms relative to each other. Analysis of restriction enhancing or abrogating mutations in the L2 region reveal that restriction defective mutants are unable to undergo dynamic conformational changes and do not assume compact, alpha-helical conformations in the L2 region. These data suggest a model in which conformational changes in the L2 region mediate displacement of CA bound SPRY domains to induce the destabilization of assembled capsid during restriction.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/immunology , HIV Infections/immunology , HIV-1/physiology , Macaca mulatta/immunology , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Cell Line , Dimerization , Disease Models, Animal , HIV Infections/genetics , HIV Infections/virology , HIV-1/genetics , Humans , Macaca mulatta/genetics , Macaca mulatta/virology , Mutation , Protein ConformationABSTRACT
The cellular factor TRIM5α inhibits infection by numerous retroviruses in a species-specific manner. The TRIM5α protein from rhesus macaques (rhTRIM5α) restricts infection by HIV-1 while human TRIM5α (huTRIM5α) restricts infection by murine leukemia virus (MLV). In owl monkeys a related protein TRIM-Cyp restricts HIV-1 infection. Several models have been proposed for retroviral restriction by TRIM5 proteins (TRIM5α and TRIM-Cyp). These models collectively suggest that TRIM5 proteins mediate restriction by directly binding to specific determinants in the viral capsid. Through their ability to self-associate TRIM5 proteins compartmentalize the viral capsid core and mediate its abortive disassembly via a poorly understood mechanism that is sensitive to proteasome inhibitors. In this review, we discuss TRIM5-mediated restriction in detail. We also discuss how polymorphisms within human and rhesus macaque populations have been demonstrated to affect disease progression of immunodeficiency viruses in these species.
Subject(s)
Capsid/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Retroviridae/growth & development , Animals , Antiviral Restriction Factors , HIV-1 , Humans , Leukemia Virus, Murine/metabolism , Polymorphism, Genetic/genetics , Tripartite Motif Proteins , Ubiquitin-Protein LigasesABSTRACT
The intracellular restriction factor TRIM5alpha, inhibits infection by numerous retroviruses in a species-specific manner. The best characterized example of this restriction is the TRIM5alpha protein from rhesus macaques (rhTRIM5alpha), which potently inhibits HIV-1 infection. TRIM5alpha localizes to cytoplasmic assemblies of protein referred to as cytoplasmic bodies, though the role that these bodies play in retroviral restriction is unclear. We employed a series of truncation mutants to identify a discrete region, located within the Linker2 region connecting the coiled-coil and B30.2/PRYSPRY domains of TRIM5alpha, which is required for cytoplasmic body localization. Deletion of this region in the context of full-length rhTRIM5alpha abrogates cytoplasmic body localization. Alanine mutagenesis of the residues in this region identifies two stretches of amino acids that are required for both cytoplasmic body localization and retroviral restriction. This work suggests that the determinants that mediate TRIM5alpha localization to cytoplasmic bodies play a requisite role in retroviral restriction.