Human Immunodeficiency Virus Type 1 Assembly Research Articles

Get in and get out: Remodeling of the cellular actin cytoskeleton upon HIV-1 infection.

The human immunodeficiency virus type 1 (HIV-1) is an intracellular pathogen whose replication cycle strictly depends on the host cell molecular machinery. HIV-1 crosses twice the plasma membrane, to get in and to get out of the cell. Therefore, the first and the last line of intracellular component encountered by the virus is the cortical actin network. Here, we review the role of actin and actin-related proteins in HIV-1 entry, assembly, budding, and release. We first highlight the mechanisms controlling actin polymerization at the entry site that promote the clustering of HIV-1 receptors, a crucial step for the virus to fuse with the plasma membrane. Then, we describe how actin is transiently depolymerized locally to allow the capsid to cross the actin cortex, before migrating towards the nucleus. Finally, we review the role of several actin-binding proteins in actin remodeling events required for membrane deformation and curvature at the viral assembly site as well as for virus release. Strikingly, it appears that common actin-regulating pathways are involved in viral entry and exit. However, while the role of actin remodeling during entry is well understood, this is not the case during exit. We discuss remaining challenges regarding the actin-dependent mechanisms involved in HIV-1 entry and exit, and how they could be overcome.

A Background Assessable and Correctable Bimolecular Fluorescence Complementation System for Nanoscopic Single-Molecule Imaging of Intracellular Protein-Protein Interactions.

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein-protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level. While it is thought that expressing the fusion proteins at a low level can remove false positives without impacting specific signals, there has been no effective strategy to test this possibility. Here, we present a system capable of assessing and removing BiFC false positives, termed Background Assessable and Correctable-BiFC (BAC-BiFC), in which one of the split FP fragments is fused with an optically distinct FP that serves as a reference marker, and the single-cell fluorescence ratio of the BiFC signal to the reference signal is used to gauge an optimal transfection condition. We showed that when BAC-BiFC is designed to image PPIs regulating Human Immunodeficiency Virus type 1 (HIV-1) assembly, the fluorescence ratio could decrease with decreasing probe quantity, and ratios approaching the limit of detection could allow physiologically relevant characterization of the assembly process on the nanoscale by single-molecule localization microscopy (SMLM). With much improved clarity, previously undescribed features of HIV-1 assembly were revealed.

Exploring the Morphological Landscape of Self-Assembled Hiv-1 Proteins from a Coarse-Grained Perspective

The self-assembly of structural proteins is central to the function of viruses. In the case of human immunodeficiency virus type-1 (HIV-1), the capsid-spacer peptide 1 (CA-SP1) domains are responsible for protein oligomerization. Under different in vitro and in vivo conditions, CA-SP1 is known to assemble into a diverse array of morphological states, including hexameric tubules, spheres, and cones; the latter two represent the physiological immature and mature virus, respectively. However, it remains unclear how the driving forces that lead to different morphological states are encoded in the CA-SP1 protein. To investigate HIV-1 assembly, our strategy is to systematically derive coarse-grained (CG) protein models in order to access experimentally-relevant spatiotemporal scales that are inaccessible to all-atom molecular dynamics (MD) simulations. Our CGMD simulations reveal that protein-protein association occurs across multiple, weak-affinity binding interfaces that are exposed in rare configurational states. We show that cofactors, salt, and membrane substrates perturb the population and affinity of pertinent protein interfaces, and consequently regulate the resultant morphology of assembled proteins. Our simulations suggest mechanisms by which viruses can simultaneously avoid kinetic traps (e.g. misaggregation) and evade immune responses. We conclude with potential directions for antiretroviral therapies.

Modeling of the HIV-1 Life Cycle in Productively Infected Cells to Predict Novel Therapeutic Targets.

There are many studies that model the within-host population dynamics of Human Immunodeficiency Virus Type 1 (HIV-1) infection. However, the within-infected-cell replication of HIV-1 remains to be not comprehensively addressed. There exist rather few quantitative models describing the regulation of the HIV-1 life cycle at the intracellular level. In treatment of HIV-1 infection, there remain issues related to side-effects and drug-resistance that require further search “...for new and better drugs, ideally targeting multiple independent steps in the HIV-1 replication cycle” (as highlighted recently by Tedbury & Freed, The Future of HIV-1 Therapeutics, 2015). High-resolution mathematical models of HIV-1 growth in infected cells provide an additional analytical tool in identifying novel drug targets. We formulate a high-dimensional model describing the biochemical reactions underlying the replication of HIV-1 in target cells. The model considers a nonlinear regulation of the transcription of HIV-1 mediated by Tat and the Rev-dependent transport of fully spliced and singly spliced transcripts from the nucleus to the cytoplasm. The model is calibrated using available information on the kinetics of various stages of HIV-1 replication. The sensitivity analysis of the model is performed to rank the biochemical processes of HIV-1 replication with respect to their impact on the net production of virions by one actively infected cell. The ranking of the sensitivity factors provides a quantitative basis for identifying novel targets for antiviral therapy. Our analysis suggests that HIV-1 assembly depending on Gag and Tat-Rev regulation of transcription and mRNA distribution present two most critical stages in HIV-1 replication that can be targeted to effectively control virus production. These processes are not covered by current antiretroviral treatments.

Characterizing the Protein/RNA Interactions in the Initial Events of HIV-1 Assembly

Transcription of the Human Immunodeficiency Virus type 1 (HIV-1) proviral DNA results in viral RNA (vRNA) with its monomeric form translated into the Gag polyprotein and its dimeric form selectively packaged into progeny viruses. vRNA constitutes less than 1% of cellular RNAs, yet the nucleocapsid (NC) region of Gag specifically recognizes the 5’-untranslated region (5’-UTR) of dimeric vRNA for effective packaging. In this study, we seek to biochemically characterize the protein/RNA interactions that mediate selective packaging of vRNA during the initial events of HIV-1 assembly. We have identified 20 NC binding sites on the dimeric 5’-UTR Core Encapsidation Signal (CES). Our crosslinking data showed binding of Gag derivatives to CES results in the formation of higher-ordered protein oligomers including hexamers. Negative stain Electron Microscopy (EM) further revealed these hexamers have a ring-like structure with a size similar to the capsid (CA) hexamer formed during viral assembly. To study how Gag interacts with the CES, we isolated a single stable hexamer by fusing a hexameric scaffolding protein, CCHex, to NC. Gel shift data reveals this CCHex-NC protein forms a 1:1 protein:RNA complex with CES. Competition binding assay shows CCHex-NC preferentially binds to dimeric CES. We plan to further elucidate the atomic structure of this protein/RNA complex using cryo-EM. Our results suggest clustering of Gag on the 5’-UTR increases the local protein concentration to promote the formation of Gag hexamers, which serves as the nucleation site to initiate selective genome packaging and viral assembly.

Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P2 has been of particular interest since PI(4,5)P2 is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP2 in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP2, but also of PIP3, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP2 molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP2 at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP2 concentration in this microdomain. These results are consistent with our previous observation that PIP2 is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.

Multiple, Switchable Protein:RNA Interactions Regulate Human Immunodeficiency Virus Type 1 Assembly.

Human immunodeficiency virus type 1 (HIV-1) particle assembly requires several protein:RNA interactions that vary widely in their character, from specific recognition of highly conserved and structured viral RNA elements to less specific interactions with variable RNA sequences. Genetic, biochemical, biophysical, and structural studies have illuminated how virion morphogenesis is accompanied by dramatic changes in the interactions among the protein and RNA virion components. The 5' leader RNA element drives RNA recognition by Gag upon initiation of HIV-1 assembly and can assume variable conformations that influence translation, dimerization, and Gag recognition. As Gag multimerizes on the plasma membrane, forming immature particles, its RNA binding specificity transiently changes, enabling recognition of the A-rich composition of the viral genome. Initiation of assembly may also be regulated by occlusion of the membrane binding surface of Gag by tRNA. Finally, recent work has suggested that RNA interactions with viral enzymes may activate and ensure the accuracy of virion maturation.

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules.IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies.

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.

HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites.

The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.

DHX9/RHA Binding to the PBS-Segment of the Genomic RNA during HIV-1 Assembly Bolsters Virion Infectivity

Cellular RNA-binding proteins incorporated into virions during human immunodeficiency virus type 1 (HIV-1) assembly promote the replication efficiency of progeny virions. Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA) to virions remains unclear. Here, cell-based experiments demonstrate that the truncation of segments of the HIV-1 5′-untranslated region (5′-UTR) distinct from the core encapsidation sequence eliminated virion incorporation of RHA, indicating that RHA recruitment is mediated by specific interactions with the HIV-1 5′-UTR. In agreement with biological data, isothermal titration calorimetry determined that the dimer conformation of the 5′-UTR binds one RHA molecule per RNA strand, and the interaction is independent of nucleocapsid protein binding. NMR spectra employing a deuterium-labeling approach enabled resolution of the dimeric 5′-UTR in complex with the RHA N-terminal domain. The structure of the large molecular mass complex was dependent on RHA binding to a double-stranded region of the primer binding site (PBS)-segment of the 5′-UTR. A single A-to-C substitution was sufficient to disrupt biophysical conformation and attenuate virion infectivity in cell-based assays. Taken together, our studies demonstrate the structural basis for HIV-1 genomic RNA to recruit beneficial cellular cofactor to virions. The support of progeny virion infectivity by RHA is attributable to structure-dependent binding at the PBS‐segment of the HIV-1 5′-UTR during virus assembly.

In virio SHAPE analysis of tRNA(Lys3) annealing to HIV-1 genomic RNA in wild type and protease-deficient virus.

BackgroundtRNALys3 annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNALys3 annealing to viral RNA occurs in two steps. First, Gag promotes annealing of tRNALys3 to the viral RNA during cytoplasmic HIV-1 assembly. Second, mature nucleocapsid (NCp7), produced from the processing of Gag by viral protease during viral budding from the cell, remodels the annealed complex to form a more stable interaction between the viral RNA and tRNALys3, resulting in a more tightly bound and efficient primer for reverse transcription.ResultsIn this report, we have used in virio SHAPE analysis of both the 5´-untranslated region in HIV-1 RNA and the annealed tRNALys3 to determine structural differences of the annealed complex that occur between protease-negative (Pr-) and wild type viruses. Our results indicate that the weaker binding of tRNALys3 annealed by Gag in Pr- virions reflects both missing interactions of tRNALys3 with viral RNA regions in the upper PBS stem, and a weaker interaction with the internal stem-loop found within the unannealed primer binding site in viral RNA.ConclusionsWe propose secondary structure models for the tRNALys3/viral RNA annealed complexes in PR- and wild type viruses that support the two-step annealing model by showing that Gag promotes a partial annealing of tRNALys3 to HIV-1 viral RNA, followed by a more complete annealing by NCp7.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0171-7) contains supplementary material, which is available to authorized users.

Editorial: Role of lipids in virus assembly.

Editorial: Role of lipids in virus assembly.

Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.

Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

Comprehensive Mutational Analysis Reveals p6 Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.

The HIV-1 matrix protein does not interact directly with the protein interactive domain of AP-3δ

During the late phase of the Human Immunodeficiency Virus Type-1 (HIV-1) replication cycle, viral Gag proteins and the intact RNA genome are trafficked to specific sub-cellular membranes where virus assembly and budding occurs. Targeting to the plasma membranes of T cells and macrophages is mediated by interactions between the N-terminal matrix (MA) domain of Gag and cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] molecules. However, in macrophages and dendritic cells, a subset of Gag proteins appears to be targeted to tetraspanin enriched viral compartments, a process that appears to be mediated by MA interactions with the Delta subunit of the cellular Adaptor Protein AP-3 (AP-3δ). We cloned, overexpressed and purified the protein interactive domain of AP-3δ and probed for MA binding by NMR. Unexpectedly, no evidence of binding was observed in these in vitro experiments, even at relatively high protein concentrations (200μM), suggesting that AP-3δ plays an alternative role in HIV-1 assembly.

Evidence of a Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Machinery in HIV-1 Assembly and Release

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.

Phosphoinositides direct equine infectious anemia virus gag trafficking and release.

Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.

A simple fluorescence based assay for quantification of human immunodeficiency virus particle release

BackgroundThe assembly and release of human immunodeficiency virus (HIV) particles from infected cells represent attractive, but not yet exploited targets for antiretroviral therapy. The availability of simple methods to measure the efficiency of these replication steps in tissue culture would facilitate the identification of host factors essential for these processes as well as the screening for lead compounds acting as specific inhibitors of particle formation. We describe here the development of a rapid cell based assay for quantification of human immunodeficiency virus type 1 (HIV-1) particle assembly and/or release.ResultsUsing a fluorescently labelled HIV-derivative, which carries an eYFP domain within the main viral structural protein Gag in the complete viral protein context, the release of virus like particles could be monitored by directly measuring the fluorescence intensity of the tissue culture supernatant. Intracellular Gag was quantitated in parallel by direct fluorescence analysis of cell lysates, allowing us to normalize for Gag expression efficiency. The assay was validated by comparison with p24 capsid ELISA measurements, a standard method for quantifying HIV-1 particles. Optimization of conditions allowed the robust detection of particle amounts corresponding to 50 ng p24/ml in medium by fluorescence spectroscopy. Further adaptation to a multi-well format rendered the assay suitable for medium or high throughput screening of siRNA libraries to identify host cell factors involved in late stages of HIV replication, as well as for random screening approaches to search for potential inhibitors of HIV-1 assembly or release.ConclusionsThe fast and simple fluorescence based quantification of HIV particle release yielded reproducible results which were comparable to the well established ELISA measurements, while in addition allowing the parallel determination of intracellular Gag expression. The protocols described here can be used for screening of siRNA libraries or chemical compounds, respectively, for inhibition of HIV in a 96-well format.

Structural Convergence between Cryo-EM and NMR Reveals Intersubunit Interactions Critical for HIV-1 Capsid Function

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.