HIV-1 adaptation studies reveal a novel Env-mediated homeostasis mechanism for evading lethal hypermutation by APOBEC3G.

HIV-1 replication normally requires Vif-mediated neutralization of APOBEC3 antiviral enzymes. Viruses lacking Vif succumb to deamination-dependent and -independent restriction processes. Here, HIV-1 adaptation studies were leveraged to ask whether viruses with an irreparable vif deletion could develop resistance to restrictive levels of APOBEC3G. Several resistant viruses were recovered with multiple amino acid substitutions in Env, and these changes alone are sufficient to protect Vif-null viruses from APOBEC3G-dependent restriction in T cell lines. Env adaptations cause decreased fusogenicity, which results in higher levels of Gag-Pol packaging. Increased concentrations of packaged Pol in turn enable faster virus DNA replication and protection from APOBEC3G-mediated hypermutation of viral replication intermediates. Taken together, these studies reveal that a moderate decrease in one essential viral activity, namely Env-mediated fusogenicity, enables the virus to change other activities, here, Gag-Pol packaging during particle production, and thereby escape restriction by the antiviral factor APOBEC3G. We propose a new paradigm in which alterations in viral homeostasis, through compensatory small changes, constitute a general mechanism used by HIV-1 and other viral pathogens to escape innate antiviral responses and other inhibitions including antiviral drugs.

Vpu is the main determinant for tetraspanin downregulation in HIV-1-infected cells.

UNLABELLED: Tetraspanins constitute a family of cellular proteins that organize various membrane-based processes. Several members of this family, including CD81, are actively recruited by HIV-1 Gag to viral assembly and release sites. Despite their enrichment at viral exit sites, the overall levels of tetraspanins are decreased in HIV-1-infected cells. Here, we identify Vpu as the main viral determinant for tetraspanin downregulation. We also show that reduction of CD81 levels by Vpu is not a by-product of CD4 or BST-2/tetherin elimination from the surfaces of infected cells and likely occurs through an interaction between Vpu and CD81. Finally, we document that Vpu-mediated downregulation of CD81 from the surfaces of infected T cells can contribute to preserving the infectiousness of viral particles, thus revealing a novel Vpu function that promotes virus propagation by modulating the host cell environment. IMPORTANCE: The HIV-1 accessory protein Vpu has previously been shown to downregulate various host cell factors, thus helping the virus to overcome restriction barriers, evade immune attack, and maintain the infectivity of viral particles. Our study identifies tetraspanins as an additional group of host factors whose expression at the surfaces of infected cells is lowered by Vpu. While the downregulation of these integral membrane proteins, including CD81 and CD82, likely affects more than one function of HIV-1-infected cells, we document that Vpu-mediated lowering of CD81 levels in viral particles can be critical to maintaining their infectiousness.

CD82 restrains pathological angiogenesis by altering lipid raft clustering and CD44 trafficking in endothelial cells.

BACKGROUND: Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. METHODS AND RESULTS: Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. CONCLUSIONS: Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.

Ezrin is a component of the HIV-1 virological presynapse and contributes to the inhibition of cell-cell fusion.

UNLABELLED: During cell-to-cell transmission of HIV-1, viral and cellular proteins transiently accumulate at the contact zone between infected (producer) and uninfected (target) cells, forming the virological synapse. Rearrangements of the cytoskeleton in producer and target cells are required for proper targeting of viral and cellular components during synapse formation, yet little is known about how these processes are regulated, particularly within the producer cell. Since ezrin-radixin-moesin (ERM) proteins connect F-actin with integral and peripheral membrane proteins, are incorporated into virions, and interact with cellular components of the virological presynapse, we hypothesized that they play roles during the late stage of HIV-1 replication. Here we document that phosphorylated (i.e., active) ezrin specifically accumulates at the HIV-1 presynapse in T cell lines and primary CD4(+) lymphocytes. To investigate whether ezrin supports virus transmission, we sought to ablate ezrin expression in producer cells. While cells did not tolerate a complete knockdown of ezrin, even a modest reduction of ezrin expression (~50%) in HIV-1-producing cells led to the release of particles with impaired infectivity. Further, when cocultured with uninfected target cells, ezrin-knockdown producer cells displayed reduced accumulation of the tetraspanin CD81 at the synapse and fused more readily with target cells, thus forming syncytia. Such an outcome likely is not optimal for virus dissemination, as evidenced by the fact that, in vivo, only relatively few infected cells form syncytia. Thus, ezrin likely helps secure efficient virus spread not only by enhancing virion infectivity but also by preventing excessive membrane fusion at the virological synapse. IMPORTANCE: While viruses, in principal, can propagate through successions of syncytia, HIV-1-infected cells in the majority of cases do not fuse with potential target cells during viral transmission. This mode of spread is coresponsible for key features of HIV-1 pathogenesis, including killing of bystander cells and establishment of latently infected T lymphocytes. Here we identify the ERM protein family member ezrin as a cellular factor that contributes to the inhibition of cell-cell fusion and thus to suppressing excessive syncytium formation. Our analyses further suggest that ezrin, which connects integral membrane proteins with actin, functions in concert with CD81, a member of the tetraspanin family of proteins. Additional evidence, documented here and elsewhere, suggests that ezrin and CD81 cooperate to prevent cytoskeleton rearrangements that need to take place during the fusion of cellular membranes.

Clustering and mobility of HIV-1 Env at viral assembly sites predict its propensity to induce cell-cell fusion.

HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells.

Migrate3D: Software for simplified post-tracking analysis of 3D and 2D cell migration data.

Migrate3D is a cell migration analysis tool whose purpose is to computationally process positional cell tracking data generated via other image acquisition/analysis software and generate biologically meaningful results. The functionalities of Migrate3D include step-based calculations of each cell track, single-cell-level summary statistics, mean squared displacement analysis, and machine learning-based evaluation of the entire dataset and subpopulations of cells found within it. The parameters calculated within Migrate3D have been previously developed and validated by other groups, and were selected to facilitate extraction of the maximum depth of information possible from input datasets. Variables are user-adjustable to enable customized analyses of diverse motility patterns and cell types, both in three-and two-dimensional timelapse data. Independent of any particular upstream image analysis or cell tracking software, Migrate3D only needs positional data over time to execute the suite of calculations. This presents a unique opportunity to standardize and streamline cell migration analysis.

HIV-1 assembly differentially alters dynamics and partitioning of tetraspanins and raft components.

Partitioning of membrane proteins into various types of microdomains is crucial for many cellular functions. Tetraspanin-enriched microdomains (TEMs) are a unique type of protein-based microdomain, clearly distinct from membrane rafts, and important for several cellular processes such as fusion, migration and signaling. Paradoxically, HIV-1 assembly/egress occurs at TEMs, yet the viral particles also incorporate raft lipids. Using different quantitative microscopy approaches, we investigated the dynamic relationship between TEMs, membrane rafts and HIV-1 exit sites, focusing mainly on the tetraspanin CD9. Our results show that clustering of CD9 correlates with multimerization of the major viral structural component, Gag, at the plasma membrane. CD9 exhibited confined behavior and reduced lateral mobility at viral assembly sites, suggesting that Gag locally traps tetraspanins. In contrast, the raft lipid GM1 and the raft-associated protein CD55, while also recruited to assembly/budding sites, were only transiently trapped in these membrane areas. CD9 recruitment and confinement were found to be partially dependent on cholesterol, while those of CD55 were completely dependent on cholesterol. Importantly, our findings support the emerging concept that cellular and viral components, instead of clustering at preexisting microdomain platforms, direct the formation of distinct domains for the execution of specific functions.

Tetraspanin functions during HIV-1 and influenza virus replication.

By virtue of their multiple interactions with partner proteins and due to their strong propensity to multimerize, tetraspanins create scaffolds in membranes, recruiting or excluding specific proteins needed for particular cellular processes. We and others have shown that (i) HIV-1 assembles at, and buds through, membrane areas that are enriched in tetraspanins CD9, CD63, CD81 and CD82, and (ii) the presence of these proteins at exit sites and in viral particles inhibits virus-induced membrane fusion. In the present paper, I review these findings and briefly discuss the results of our ongoing investigations that are aimed at elucidating when and how tetraspanins regulate this fusion process and how such control affects virus spreading. Finally, I give a preview of studies that we have initiated more recently and which aim to delineate exactly when CD81 functions during the replication of another enveloped RNA virus: influenza virus.

Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1.

Specific spatial arrangements of proteins and lipids are central to the coordination of many biological processes. Tetraspanins have been proposed to laterally organize cellular membranes via specific associations with each other and with distinct integrins. Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane. Fluorescence and immunoelectron microscopic analyses document that the surface of HeLa cells is covered by several hundred TEMs, each extending over a few hundred nanometers and containing predominantly two or more tetraspanins. Further, we reveal that the human immunodeficiency virus type 1 (HIV-1) Gag protein, which directs viral assembly and release, accumulates at surface TEMs together with the HIV-1 envelope glycoprotein. TSG101 and VPS28, components of the mammalian ESCRT1 (endosomal sorting complex required for transport), which is part of the cellular extravesiculation machinery critical for HIV-1 budding, are also recruited to cell surface TEMs upon virus expression, suggesting that HIV-1 egress can be gated through these newly mapped microdomains.

CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion.

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific monoclonal antibody, inhibits the release of HIV-1 and canine distemper virus (CDV)- but not measles virus (MV)-induced cell-cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas. High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins beta(1)-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the fusion machinery to cell contact areas.

Formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells.

In vitro propagation studies have established that human immunodeficiency virus type 1 (HIV-1) is most efficiently transmitted at the virological synapse that forms between producer and target cells. Despite the presence of the viral envelope glycoprotein (Env) and CD4 and chemokine receptors at the respective surfaces, producer and target cells usually do not fuse with each other but disengage after the viral particles have been delivered, consistent with the idea that syncytia, at least in vitro, are not required for HIV-1 spread. Here, we tested whether tetraspanins, which are well known regulators of cellular membrane fusion processes that are enriched at HIV-1 exit sites, regulate syncytium formation. We found that overexpression of tetraspanins in producer cells leads to reduced syncytium formation, while downregulation has the opposite effect. Further, we document that repression of Env-induced cell-cell fusion by tetraspanins depends on the presence of viral Gag, and we demonstrate that fusion repression requires the recruitment of Env by Gag to tetraspanin-enriched microdomains (TEMs). However, sensitivity to fusion repression by tetraspanins varied for different viral strains, despite comparable recruitment of their Envs to TEMs. Overall, these data establish tetraspanins as negative regulators of HIV-1-induced cell-cell fusion, and they start delineating the requirements for this regulation.

The roles of tetraspanins in HIV-1 replication.

Tetraspanins are small integral membrane proteins that are known to control a variety of cellular processes, including signaling, migration and cell-cell fusion. Research over the past few years established that they are also regulators of various steps in the HIV-1 replication cycle, but the mechanisms through which these proteins either enhance or repress virus spread remain largely unknown.

Tetraspanins regulate cell-to-cell transmission of HIV-1.

BACKGROUND: The presence of the tetraspanins CD9, CD63, CD81 and CD82 at HIV-1 budding sites, at the virological synapse (VS), and their enrichment in HIV-1 virions has been well-documented, but it remained unclear if these proteins play a role in the late phase of the viral replication cycle. Here we used overexpression and knockdown approaches to address this question. RESULTS: Neither ablation of CD9, CD63 and/or CD81, nor overexpression of these tetraspanins was found to affect the efficiency of virus release. However, confirming recently reported data, tetraspanin overexpression in virus-producing cells resulted in the release of virions with substantially reduced infectivity. We also investigated the roles of these tetraspanins in cell-to-cell transmission of HIV-1. Overexpression of CD9 and CD63 led to reduced cell-to-cell transmission of this virus. Interestingly, in knockdown experiments we found that ablation of CD63, CD9 and/or CD81 had no effect on cell-free infectivity. However, knockdown of CD81, but not CD9 and CD63, enhanced productive particle transmission to target cells, suggesting additional roles for tetraspanins in the transmission process. Finally, tetraspanins were found to be downregulated in HIV-1-infected T lymphocytes, suggesting that HIV-1 modulates the levels of these proteins in order to maximize the efficiency of its transmission within the host. CONCLUSION: Altogether, these results establish an active role of tetraspanins in HIV-1 producer cells.

EWI-2 Inhibits Cell-Cell Fusion at the HIV-1 Virological Presynapse.

Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell-cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell-cell fusion. Using quantitative fluorescence microscopy, shRNA knockdowns, and cell-cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also find that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell-cell fusion and provides novel insight into how syncytia are prevented from fusing indefinitely.

NEIL3 Repairs Telomere Damage during S Phase to Secure Chromosome Segregation at Mitosis.

Oxidative damage to telomere DNA compromises telomere integrity. We recently reported that the DNA glycosylase NEIL3 preferentially repairs oxidative lesions in telomere sequences in vitro. Here, we show that loss of NEIL3 causes anaphase DNA bridging because of telomere dysfunction. NEIL3 expression increases during S phase and reaches maximal levels in late S/G2. NEIL3 co-localizes with TRF2 and associates with telomeres during S phase, and this association increases upon oxidative stress. Mechanistic studies reveal that NEIL3 binds to single-stranded DNA via its intrinsically disordered C terminus in a telomere-sequence-independent manner. Moreover, NEIL3 is recruited to telomeres through its interaction with TRF1, and this interaction enhances the enzymatic activity of purified NEIL3. Finally, we show that NEIL3 interacts with AP Endonuclease 1 (APE1) and the long-patch base excision repair proteins PCNA and FEN1. Taken together, we propose that NEIL3 protects genome stability through targeted repair of oxidative damage in telomeres during S/G2 phase.

HIV-1-Induced Small T Cell Syncytia Can Transfer Virus Particles to Target Cells through Transient Contacts.

HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing "fusion sinks" are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis.

Evidence showing that tetraspanins inhibit HIV-1-induced cell-cell fusion at a post-hemifusion stage.

Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening.

The intracellular cargo receptor ERGIC-53 is required for the production of infectious arenavirus, coronavirus, and filovirus particles.

Arenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families.

For HIV, it's never too late to grow up.

Rather than being separated by discrete boundaries, the different phases of viral replication cycles partially overlap one another. In this issue, Dale et al. (2011) describe a remarkable example of this phenomenon: HIV-1 matures, and thus becomes infectious, but only after it has already started entering target cells.