Katy Janvier

Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes

Aquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to specific membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also specifically expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane trafficking remain largely unknown. Here, we show that two independent C‐terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine‐based motif; the other is encoded by a di‐leucine‐like motif. We found that the tyrosine‐based basolateral sorting signal also determines AQP4 clathrin‐dependent endocytosis through direct interaction with the μ subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in μ subunit interaction changes AP2 adaptor association to AP3. We found that the stress‐induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4–μ3A interaction and enhancing AQP4–lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression.

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.

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.

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.

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.

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

ABSTRACT 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 β-TrCP1 and β-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 β-TrCP proteins. Altogether, our results provide further understanding of the mechanisms underlying BST2 turnover in cells. Highlighted Article: We identify two E3 ubiquitin ligases, NEDD4 and MARCH8, as regulators of BST2 (tetherin) – a protein that restricts viral release; we thus provide further understanding of the mechanisms underlying BST2 turnover in cells.

Role of Rab proteins in the formation of HIV-1 particles

The production of HIV-1 particles during the late steps of viral multiplication cycle corresponds to the assembly, the budding and the release of infectious viral particles in the extracellular medium. These steps require the integrity of two viral structural components: the Gag precursor, essential for HIV assembly and budding, and the envelope glycoprotein (Env) which confers the infectivity to the virus after its incorporation into the viral Gag particles. n nThe last steps of the viral cycle involve a series of molecular and cellular events based on interactions between viral proteins and cellular proteins that are implicated in the vesicular intracellular trafficking, such as the ESCRT machinery, the AP clathrin adaptors or the cellular cofactor TIP47. Although several interactions between viral proteins and cellular cofactors have been described, the identification of new cellular partners is crucial in order to understand the complex interplay between HIV-1 and the host cell during the late steps of the HIV cycle. To characterize new cofactors involved in HIV assembly, we decided to assess the role of Rab proteins, key regulators of vesicular intracellular trafficking in the infectious viral particles production. n nFor this purpose, we developed several virological tests based on the specific interference RNA targeting Rab proteins (Rab1, Rab4, Rab5, Rab6, Rab7, Rab8, Rab9 and Rab11). We show that Rab7 plays a major role on HIV-1 replication. We observed that HIV-1 (NL4-3 strain) failed to propagate in the reporter cells (HeLa P4R5 - stably expressing CD4 and CCR5) upon siRNA-induced depletion of Rab7. Using a single cycle infection assay, we showed that Rab7 depletion causes a decrease of HIV-1 release from the producer cells. Moreover, Rab7 depletion modified Gag processing and the infectivity of the produced particles, a defect usually observed upon TSG101 depletion. n nAltogether, our data highlight a key role of Rab7 in the morphogenesis of new infectious HIV particles. This work is funded by ANRS, SIDACTION, ANR-07-JCJC-0102 programs and is part of the activities of the HIV-ACE research network (HEALTH-F3-2008-201095) supported by a grant of the European Commission, within the Priority 1 Health work programme of the 7th Framework Programme of the EU.

Response to Luca L Fava and colleagues.

We read with great interest the letter of Fava and colleagues, who challenge the conclusions of our article published in EMBO Reports (2013) [1]. Our work described a role for Beclin‐1 in chromosome congression and outer kinetochore assembly. We demonstrated that this unexpected role of Beclin‐1 is independent of its association with the PI3K‐III complex and its well‐documented role in autophagy. Our findings were based on knockdown of Beclin‐1 using two different siRNAs and on rescue experiments. Additional biochemical and cellular biology experiments with native proteins also suggested a functional role for Beclin‐1 in outer kinetochore assembly.nnIn this issue of EMBO Reports , Fava and colleagues suggest that Beclin‐1 is dispensable for chromosome congression. They performed a set of experiments with two additional siRNAs targeting Beclin‐1. One of them efficiently knocks down Beclin‐1 at the protein level (siRNA Becn1‐II); the second one only partially depletes Beclin‐1 (siRNA Becn1‐I). They report in their study that, in contrast to what we described, neither …

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

Macrophages are, along with T CD4+lymphocytes, the major targets of HIV-1 infection and play a key role in viral pathogenesis as a significant reservoir. Identification of the cellular cofactors involved in the production of infectious HIV-1 from macrophages is thus crucial. Nevertheless molecular mechanisms allowing the production of infectious particles from macrophages are not entirely deciphered. In this study, we investigated the role of the cellular cofactor TIP47 in HIV-1 morphogenesis in primary macrophages. Our data show that TIP47 is essential for HIV-1 infectivity and propagation. Mutations in HIV-1 Gag or Envelope (Env) proteins, which abolish interaction with TIP47, impair HIV-1 propagation and infectivity preventing colocalization of Gag and Env, and thereby inhibiting Env incorporation into virions. Whereas disruption of Gag-TIP47 interaction increases slightly Gag particles production, impaired Env-TIP47 binding reduces Gag particles release and, strikingly, induces their retention in the viral assembly compartments. Thus, as in T lymphocytes, TIP47 is critical for the efficient release of infectious HIV-1 particles from the assembly compartment of primary macrophages, making TIP47 a new potential therapeutic target for limiting HIV-1 infectivity and spreading. This work is funded by ANRS, SIDACTION, ANR-07-JCJC-0102 programs and is part of the activities of the HIV-ACE research network (HEALTH-F3-2008-201095) supported by a grant of the European Commission, within the Priority 1 Health work programme of the 7th Framework Programme of the EU.