ATG5 selectively engages virus-tethered BST2/tetherin in an LC3C-associated pathway.

Bone marrow stromal antigen 2 (BST2)/tetherin is a restriction factor that reduces HIV-1 dissemination by tethering virus at the cell surface. BST2 also acts as a sensor of HIV-1 budding, establishing a cellular antiviral state. The HIV-1 Vpu protein antagonizes BST2 antiviral functions via multiple mechanisms, including the subversion of an LC3C-associated pathway, a key cell intrinsic antimicrobial mechanism. Here, we describe the first step of this viral-induced LC3C-associated process. This process is initiated at the plasma membrane through the recognition and internalization of virus-tethered BST2 by ATG5, an autophagy protein. ATG5 and BST2 assemble as a complex, independently of the viral protein Vpu and ahead of the recruitment of the ATG protein LC3C. The conjugation of ATG5 with ATG12 is dispensable for this interaction. ATG5 recognizes cysteine-linked homodimerized BST2 and specifically engages phosphorylated BST2 tethering viruses at the plasma membrane, in an LC3C-associated pathway. We also found that this LC3C-associated pathway is used by Vpu to attenuate the inflammatory responses mediated by virion retention. Overall, we highlight that by targeting BST2 tethering viruses, ATG5 acts as a signaling scaffold to trigger an LC3C-associated pathway induced by HIV-1 infection.

Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages.

HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+ T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu's ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64 and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCE HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu's ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.

Characterization of E3 ligases involved in lysosomal sorting of the HIV-1 restriction factor BST2.

The cellular protein BST2 (also known as tetherin) acts as a major intrinsic antiviral protein that prevents the release of enveloped viruses by trapping nascent viral particles at the surface of infected cells. Viruses have evolved specific strategies to displace BST2 from viral budding sites in order to promote virus egress. In HIV-1, the accessory protein Vpu counters BST2 antiviral activity and promotes sorting of BST2 for lysosomal degradation. Vpu increases polyubiquitylation of BST2, a post-translation modification required for Vpu-induced BST2 downregulation, through recruitment of the E3 ligase complex SCF adaptors ß-TrCP1 and ß-TrCP2 (two isoforms encoded by BTRC and FBXW11, respectively). Herein, we further investigate the role of the ubiquitylation machinery in the lysosomal sorting of BST2. Using a small siRNA screen, we highlighted two additional regulators of BST2 constitutive ubiquitylation and sorting to the lysosomes: the E3 ubiquitin ligases NEDD4 and MARCH8. Interestingly, Vpu does not hijack the cellular machinery that is constitutively involved in BST2 ubiquitylation to sort BST2 for degradation in the lysosomes but instead promotes the recognition of BST2 by ß-TrCP proteins. Altogether, our results provide further understanding of the mechanisms underlying BST2 turnover in cells.

LC3C Contributes to Vpu-Mediated Antagonism of BST2/Tetherin Restriction on HIV-1 Release through a Non-canonical Autophagy Pathway.

BST2 (bone marrow stromal antigen 2)/tetherin is a restriction factor of enveloped viruses, which blocks the release of viral particles. HIV-1 encodes proteins that antagonize this innate barrier, including the accessory protein Vpu. Here, we investigate whether the autophagy pathway and/or ATG proteins are hijacked by HIV-1 Vpu to circumvent BST2 restriction of viral release. We report that BST2 and Vpu are present in LC3-positive compartments. We found that Vpu selectively interacts with the ATG8 ortholog LC3C through the Vpu L63VEM66 sequence. This sequence is required for Vpu to antagonize BST2 restriction. LC3C expression favors the removal of BST2 from the HIV-1 budding site, and thus HIV-1 release in BST2-expressing cells. Additionally, ATG5 and beclin 1/ATG6, but not all the components of the autophagy pathway, act with LC3C to facilitate Vpu antagonism of BST2 restriction. Altogether, our data support the view that a non-canonical autophagy pathway reminiscent of LC3-associated phagocytosis contributes to Vpu counteraction of BST2 restriction.

Mechanisms underlying HIV-1 Vpu-mediated viral egress.

Viruses such as lentiviruses that are responsible for long lasting infections have to evade several levels of cellular immune mechanisms to persist and efficiently disseminate in the host. Over the past decades, much evidence has emerged regarding the major role of accessory proteins of primate lentiviruses, human immunodeficiency virus and simian immunodeficiency virus, in viral evasion from the host immune defense. This short review will provide an overview of the mechanism whereby the accessory protein Vpu contributes to this escape. Vpu is a multifunctional protein that was shown to contribute to viral egress by down-regulating several mediators of the immune system such as CD4, CD1d, NTB-A and the restriction factor BST2. The mechanisms underlying its activity are not fully characterized but rely on its ability to interfere with the host machinery regulating protein turnover and vesicular trafficking. This review will focus on our current understanding of the mechanisms whereby Vpu down-regulates CD4 and BST2 expression levels to favor viral egress.

Beclin-1 is required for chromosome congression and proper outer kinetochore assembly.

The functions of Beclin-1 in macroautophagy, tumorigenesis and cytokinesis are thought to be mediated by its association with the PI3K-III complex. Here, we describe a new role for Beclin-1 in mitotic chromosome congression that is independent of the PI3K-III complex and its role in autophagy. Beclin-1 depletion in HeLa cells leads to a significant reduction of the outer kinetochore proteins CENP-E, CENP-F and ZW10, and, consequently, the cells present severe problems in chromosome congression. Beclin-1 associates with kinetochore microtubules and forms discrete foci near the kinetochores of attached chromosomes. We show that Beclin-1 interacts directly with Zwint-1-a component of the KMN (KNL-1/Mis12/Ndc80) complex-which is essential for kinetochore-microtubule interactions. This suggests that Beclin-1 acts downstream of the KMN complex to influence the recruitment of outer kinetochore proteins and promotes accurate kinetochore anchoring to the spindle during mitosis.

Role of the endosomal ESCRT machinery in HIV-1 Vpu-induced down-regulation of BST2/tetherin.

The cellular protein "Bone marrow stromal antigen 2" (BST2 also called Tetherin, CD317, HM1.24) was identified as a major mediator of the innate immune defense against the dissemination of enveloped viruses. BST2 was shown to physically trap the de novo formed viral particles at the surface of infected cells, thereby reducing viral release. Lentiviruses have evolved specific strategies to down-regulate the expression level of BST2 from the surface of the cells and as such promote viral egress. In Human Immunodeficiency Virus-1 (HIV-1), the accessory protein Vpu counters BST2 antiviral activity. However, the cellular and molecular mechanisms involved are not fully understood. Vpu-mediated antagonism of BST2 antiviral activity seems to involve complex interplay between the viral protein and host components regulating protein turnover and vesicular trafficking. This review focuses on the interplay between Vpu and the ubiquitin/endosomal pathway in countermeasures of HIV-1 to BST2 restriction, with a particular emphasis on the "Endosomal Sorting Complexes Required for Transport" (ESCRT) machinery.

Rab7A is required for efficient production of infectious HIV-1.

Retroviruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Rab proteins regulate specific steps in intracellular membrane trafficking by recruiting tethering, docking and fusion factors, as well as the actin- and microtubule-based motor proteins that facilitate vesicle traffic. Using virological tests and RNA interference targeting Rab proteins, we demonstrate that the late endosome-associated Rab7A is required for HIV-1 propagation. Analysis of the late steps of the HIV infection cycle shows that Rab7A regulates Env processing, the incorporation of mature Env glycoproteins into viral particles and HIV-1 infectivity. We also show that siRNA-mediated Rab7A depletion induces a BST2/Tetherin phenotype on HIV-1 release. BST2/Tetherin is a restriction factor that impedes HIV-1 release by tethering mature virus particles to the plasma membrane. Our results suggest that Rab7A contributes to the mechanism by which Vpu counteracts the restriction factor BST2/Tetherin and rescues HIV-1 release. Altogether, our results highlight new roles for a major regulator of the late endocytic pathway, Rab7A, in the late stages of the HIV-1 replication cycle.

The ESCRT-0 component HRS is required for HIV-1 Vpu-mediated BST-2/tetherin down-regulation.

The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, a highly conserved set of four hetero-oligomeric protein complexes, is required for multivesicular body formation, sorting ubiquitinylated membrane proteins for lysosomal degradation, cytokinesis and the final stages of assembly of a number of enveloped viruses, including the human immunodeficiency viruses. Here, we show an additional role for the ESCRT machinery in HIV-1 release. BST-2/tetherin is a restriction factor that impedes HIV release by tethering mature virus particles to the plasma membrane. We found that HRS, a key component of the ESCRT-0 complex, promotes efficient release of HIV-1 and that siRNA-mediated HRS depletion induces a BST-2/tetherin phenotype. This activity is related to the ability of the HIV-1 Vpu protein to down-regulate BST-2/tetherin. We found that BST-2/tetherin undergoes constitutive ESCRT-dependent sorting for lysosomal degradation and that this degradation is enhanced by Vpu expression. We demonstrate that Vpu-mediated BST-2/tetherin down-modulation and degradation require HRS (ESCRT-0) function and that knock down of HRS increases cellular levels of BST-2/tetherin and restricts virus release. Furthermore, HRS co-precipitates with Vpu and BST-2. Our results provide further insight into the mechanism by which Vpu counteracts BST-2/tetherin and promotes HIV-1 dissemination, and they highlight an additional role for the ESCRT machinery in virus release.

TIP47 is required for the production of infectious HIV-1 particles from primary macrophages.

Macrophages are among the major targets of HIV-1 infection and play a key role in viral pathogenesis. Identification of the cellular cofactors involved in the production of infectious HIV-1 from macrophages is thus crucial. Here, we investigated the role of the cellular cofactor TIP47 in HIV-1 morphogenesis in primary macrophages. Using siRNA approach, we show that TIP47 is essential for HIV-1 infectivity and propagation. TIP47 silencing disrupts Gag and Env colocalization in macrophages. Moreover, mutations in HIV-1 Gag or Env, which abolish interaction with TIP47, impair HIV-1 propagation and infectivity preventing colocalization of Gag and Env, Gag and Env coimmunoprecipitation. Interestingly, disruption of Gag-TIP47 interaction by matrix mutation or TIP47 depletion also causes Gag to localize in scattered dots in the vicinity of the plasma membrane of macrophages. Therefore, TIP47 is required for the encounter between Gag and Env, and thus for the generation of infectious HIV-1 particles from primary macrophages.

Human immunodeficiency virus type 1 Nef protein targets CD4 to the multivesicular body pathway.

The Nef protein of human immunodeficiency virus type 1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. Herein, we report that Nef has the additional function of targeting CD4 to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. This targeting involves the endosomal sorting complex required for transport (ESCRT) machinery. Perturbation of this machinery does not prevent removal of CD4 from the cell surface but precludes its lysosomal degradation, indicating that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. We also show that both CD4 and Nef are ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway.

Role of the endocytic machinery in the sorting of lysosome-associated membrane proteins.

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin-and to a lesser extent the other AP complexes-are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

CD4 down-regulation by HIV-1 and simian immunodeficiency virus (SIV) Nef proteins involves both internalization and intracellular retention mechanisms.

Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.

Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity.

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.

An ear-core interaction regulates the recruitment of the AP-3 complex to membranes.

AP-3 is a heterotetrameric adaptor involved in the biogenesis of lysosome-related organelles. The function of AP-3 as an adaptor relies on its ability to bind to membranes in an Arf-dependent fashion and to recognize sorting signals in the cytosolic tails of the transmembrane cargo. Here, we report an interdomain interaction involving the ear domain of the delta subunit and the sigma3 subunit of AP-3. This interaction interferes with the binding of AP-3 to Arf but not to dileucine-based sorting signals. As a consequence, the delta-ear inhibits the recruitment of AP-3 to membranes both in vitro and in vivo and impairs the sorting of lysosomal membrane proteins. These observations suggest a new regulatory mechanism for the recruitment of AP-3 to membranes involving delta-ear-sigma3 interactions.

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes.

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.

Targeting of the human immunodeficiency virus type 1 envelope to the trans-Golgi network through binding to TIP47 is required for env incorporation into virions and infectivity.

Here, we report that human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is located mainly in the trans-Golgi network (TGN) due to determinants present in the cytoplasmic domain of the transmembrane gp41 glycoprotein (TMgp41). Internalization assays demonstrated that Env present at the cell surface returns to the TGN. We found that the cytoplasmic domain of TMgp41 binds to TIP47, a protein required for the transport of mannose-6-phosphate receptors from endosomes to the TGN. Overexpression of a mutant of TIP47 affected the transport of Env from endosomes to the TGN. Retrograde transport of Env to the TGN requires a Y(802)W(803) diaromatic motif present in the TMgp41 cytoplasmic domain. Mutation of this motif abolished both targeting to the TGN as well as interaction with TIP47. These data support the view that binding of TIP47 to HIV-1 Env facilitates its delivery to the TGN. Lastly, we show that virus mutated in the Y(802)W(803) motif is poorly infectious and presents a defect in Env incorporation, supporting a model in which retrograde transport of Env is implicated in the optimization of fully infectious HIV-1 production.

HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes.

The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.