HIV-1 Assembly Sites Research Articles

Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy.

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.

Super-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells.

The ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions.

Lipid biosensor interactions with wild type and matrix deletion HIV-1 Gag proteins

The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes. Our investigations showed marked differences in WT and ΔMA Gag colocalization with biosensors, effects on biosensor release, and association of biosensors with virus-like particles. These results demonstrate an alternative approach to the analysis of viral protein-lipid associations, and provide new data as to the lipid compositions of HIV-1 assembly sites.

Rab27a builds a platform for HIV-1

![Figure][1] Compared with a control T cell (left), knocking down Rab27a blocks the delivery of late endosomes (red) to the plasma membrane, inhibiting the recruitment of Pr55Gag (green) to HIV-1 assembly sites. The endosomal trafficking protein Rab27a supports HIV-1 replication by

Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites.

The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.

Retrovirus Glycoprotein Functionality Requires Proper Alignment of the Ectodomain and the Membrane-Proximal Cytoplasmic Tail

Nonnative viral glycoproteins, including Friend murine leukemia virus envelope (F-MLV Env) are actively recruited to HIV-1 assembly sites by an unknown mechanism. Because interactions with the lipid microenvironment at budding sites could contribute to recruitment, we examined the contribution of the hydrophobicity of the F-MLV Env membrane-spanning domain (MSD) to its incorporation into HIV-1 particles. A series of F-MLV Env mutants that added or deleted one, two, or three leucines in the MSD were constructed. All six mutants retained the ability to be incorporated into HIV-1 particles, but the -1L, -2L, -3L, +1L, and +2L mutants were not capable of producing infectious particles. Surprisingly, the +3L Env glycoprotein was able to produce infectious particles and was constitutively fusogenic. However, when the cytoplasmic tail domains (CTDs) in the Env constructs were deleted, all six of the MSD mutants were able to produce infectious particles. Further mutational analyses revealed that the first 10 amino acids of the CTD is a critical regulator of infectivity. A similar phenotype was observed in HIV-1 Env upon addition of leucines in the MSD, with +1 and +2 leucine mutations greatly reducing Env activity, but +3 leucine mutations behaving similar to the wild type. Unlike F-MLV Env (+1L and +2L), HIV-1 Env (+1L and +2L) infectivity was not restored by deletion of the CTD. We hypothesize that the CTD forms a coiled-coil that disrupts the protein's functionality if it is not in phase with the trimer interface of the ectodomain.

Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail

The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.

Roles played by capsid-dependent induction of membrane curvature and Gag-ESCRT interactions in tetherin recruitment to HIV-1 assembly sites.

Tetherin/BST-2 (here called tetherin) is an antiviral protein that restricts release of diverse enveloped viruses from infected cells through physically tethering virus envelope and host plasma membrane. For HIV-1, specific recruitment of tetherin to assembly sites has been observed as its colocalization with the viral structural protein Gag or its accumulation in virus particles. Because of its broad range of targets, we hypothesized that tetherin is recruited through conserved features shared among various enveloped viruses, such as lipid raft association, membrane curvature, or ESCRT dependence. We observed that reduction of cellular cholesterol does not block tetherin anti-HIV-1 function, excluding an essential role for lipid rafts. In contrast, mutations in the capsid domain of Gag, which inhibit induction of membrane curvature, prevented tetherin-Gag colocalization detectable by confocal microscopy. Disruption of Gag-ESCRT interactions also inhibited tetherin-Gag colocalization when disruption was accomplished via amino acid substitutions in late domain motifs, expression of a dominant-negative Tsg101 derivative, or small interfering RNA (siRNA)-mediated depletion of Tsg101 or Alix. However, further analyses of these conditions by quantitative superresolution localization microscopy revealed that Gag-tetherin coclustering is significantly reduced but persists at intermediate levels. Notably, this residual tetherin recruitment was still sufficient for the full restriction of HIV-1 release. Unlike the late domain mutants, the capsid mutants defective in inducing membrane curvature showed little or no coclustering with tetherin in superresolution analyses. These results support a model in which both Gag-induced membrane curvature and Gag-ESCRT interactions promote tetherin recruitment, but the recruitment level achieved by the former is sufficient for full restriction.

Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolution

We apply single-molecule super-resolution microscopy and coordinate-based cluster analysis to extract information on the distribution and on the morphology and size of clusters of the human immunodeficiency virus (HIV-1) Gag polyprotein in fixed cells. Three different patterns of Gag distribution could be distinguished. A major type of assembly observed was in accordance with previous electron microscopy analyses revealing ~140nm-sized HIV-1 buds at the plasma membrane of virus-producing cells. The distribution of Gag molecules in the 2D projection at these sites was consistent with a semi-spherical 3D assembly. We compared different methods of cluster analysis and demonstrated that we can reliably distinguish different distribution patterns of the Gag polyprotein. These methods were applied to extract information on the properties of the different Gag clusters.

Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane

The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominantly at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.