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Movie 4 AVI file - 879K, Time-lapse videomicroscopy of wound healing assay using GFP-ATIP3-transiently transfected D3H2LN cells

Movie 3 AVI file - 997K, Time-lapse videomicroscopy of wound healing assay using GFP-transiently transfected D3H2LN cells

Supplementary Tables XLS file - 200K, Table SI : breast tumors and normal samples data; Table SII: MTUS1 expression level in invasive breast tumors collected at Institut Gustave Roussy; Table SIII: Time-course of metastasis formation

Movie 2 AVI file - 2424K, Time-lapse videomicroscopy of wound healing assay using GFP-ATIP3-transfected D3H2LN clone (Cl3)

Movie 5 AVI file - 6439K, Time-lapse videomicroscopy of EB1-GFP comets in siRNA-silenced HeLa cells

Supplementary Information PDF file - 115K, Supplemental materials and methods, legends to Movies and supplemental references

Movie 6 AVI file - 5618K, Time-lapse videomicroscopy of EB3-GFP comets in wild type and mCherry-ATIP3 transfected MRC5 cells

Supplementary Figures PDF file - 486K, Figure S1. Low levels of ATIP3 associate with decreased overall and relapse-free survival of the patients;Figure S2. Anti-metastatic effects of ATIP3 in vivo; Figure S3. ATIP3 inhibits breast cancer cell proliferation and migration; Figure S4. ATIP3 regulates MT dynamics; Figure S5. Characterization of ATIP3 domains

Movie 1 AVI file - 2575K, Time-lapse videomicroscopy of wound healing assay using GFP-transfected D3H2LN clone

Movie 7 AVI file - 629K, Time-lapse videomicroscopy of wound healing assay in GFP-D2-transiently transfected D3H2LN cells

The HUSH complex (composed of TASOR, MPP8 and periphilin) represses HIV-1 expression from its promoter by inducing both propagation of repressive epigenetic marks and degradation of the nascent transcript. Vpx from HIV-2, and Vpr proteins from some simian lentiviruses (SIVs), antagonize HUSH, thereby increasing proviral expression. The chromatin-remodelling MORC2 protein plays a critical role in the epigenetic silencing of host genes by HUSH. Here, we deciphered the role of MORC2 in retroviral silencing. We show that MORC2, in contrast to HUSH components, presents strong signatures of positive selection during primate evolution. Like HUSH, MORC2 represses proviral expression in two models of HIV-1 latency. However, while HUSH is degraded upon HIV-2 infection in a Vpx-dependent manner, MORC2 levels are increased, raising the question of a feedback control mechanism without HUSH. Upon infection with an HIV-1-derived virus, MORC2 and TASOR antiviral effects are interdependent. However, once the lentiviral DNA is integrated into the host genome, MORC2 may maintain the repression independently of HUSH. At the post-transcriptional level, both MORC2 and HUSH act in association with CNOT1 of the CCR4-NOT deadenylase complex and the TRAMP-like PAXT complex. Finally, MORC2, but not HUSH components, is expressed in primary quiescent CD4+ T cells. Altogether, our data highlight MORC2 as an HIV restriction factor and a chromatin remodelling protein operating both at the transcriptional and post-transcriptional levels. We speculate that MORC2 could serve as an immune gatekeeper following HUSH inactivation by Vpx and contribute to the maintenance of retroviral silencing in reservoir CD4+ T cells.Significance statementOne hurdle to HIV eradication is viral latency, which refers to the persistence of the virus in reservoir cells despite antiretroviral treatment. The HUSH complex represses HIV expression, once the viral genome is integrated into the host genome. HUSH activity on host genes depends on MORC2, a protein incriminated in the Charcot-Marie-Tooth neuronal disease. Here, we first show that MORC2 presents signs of evolutionary arms-races in primates. Furthermore, MORC2 contributes to HIV silencing in cooperation with HUSH, but also, likely without HUSH. Despite identified as a chromatin remodeler, MORC2 also works at a post-transcriptional level. Altogether, MORC2 appears as a host defense factor, which plays a role in HIV latency.

Human Immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) succeed to evade host immune defenses by using their viral auxiliary proteins to antagonize host restriction factors. HIV-2/SIVsmm Vpx is known for degrading SAMHD1, a factor impeding the reverse transcription. More recently, Vpx was also shown to counteract HUSH, a complex constituted of TASOR, MPP8 and periphilin, which blocks viral expression from the integrated viral DNA. In a classical ubiquitin ligase hijacking model, Vpx bridges the DCAF1 ubiquitin ligase substrate adaptor to SAMHD1, for subsequent ubiquitination and degradation. Here, we investigated whether the same mechanism is at stake for Vpx-mediated HUSH degradation. While we confirm that Vpx bridges SAMHD1 to DCAF1, we show that TASOR can interact with DCAF1 in the absence of Vpx. Nonetheless, this association was stabilized in the presence of Vpx, suggesting the existence of a ternary complex. The N-terminal PARP-like domain of TASOR is involved in DCAF1 binding, but not in Vpx binding. We also characterized a series of HIV-2 Vpx point mutants impaired in TASOR degradation, while still degrading SAMHD1. Vpx mutants ability to degrade TASOR correlated with their capacity to enhance HIV-1 minigenome expression as expected. Strikingly, several Vpx mutants impaired for TASOR degradation, but not for SAMHD1 degradation, had a reduced binding affinity for DCAF1, but not for TASOR. In macrophages, Vpx R34A-R42A and Vpx R42A-Q47A-V48A, strongly impaired in DCAF1, but not in TASOR binding, could not degrade TASOR, while being efficient in degrading SAMHD1. Altogether, our results highlight the central role of a robust Vpx-DCAF1 association to trigger TASOR degradation. We then propose a model in which Vpx interacts with both TASOR and DCAF1 to stabilize a TASOR-DCAF1 complex. Furthermore, our work identifies Vpx mutants enabling the study of HUSH restriction independently from SAMHD1 restriction in primary myeloid cells., Author summary Human Immunodeficiency Virus (HIV) is still a major public health issue. The understanding of the molecular battle occurring during viral infection, between HIV components and cellular antiviral factors, the so-called restriction factors, is a key determinant for new treatment development. Namely, HIV auxiliary proteins are powerful to induce the downregulation of cellular restriction factors by hijacking the Ubiquitin/proteasome pathway, in order to facilitate the completion of a well-processed HIV replication cycle. For instance, HIV-2 Vpx eases reverse transcription in myeloid cells by counteracting the SAMDH1 restriction factor. More recently, we discovered the ability of Vpx to induce the degradation of the HUSH epigenetic repressor complex to favor in turn, the expression of the provirus. In this study, we uncovered the mechanisms by which Vpx antagonizes TASOR, the core subunit of the HUSH complex. We highlighted key differences between Vpx-induced TASOR and SAMHD1 degradation. These findings will help to propose strategies to study or to target either HUSH or SAMHD1, especially in myeloid cells where SAMHD1 restriction operates.

The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin is involved in the spreading of H3K9me3 repressive marks across genes and transgenes such as ZNF encoding genes, ribosomal DNAs, LINE-1, Retrotransposons and Retroelements or the integrated HIV provirus1–5. The deposit of these repressive marks leads to heterochromatin formation and inhibits gene expression. The precise mechanisms of silencing mediated by HUSH is still poorly understood. Here, we show that TASOR depletion increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. By counteracting HUSH, Vpx from HIV-2 mimics TASOR depletion. With the use of a Yeast-Two-Hybrid screen, we identified new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 interact in vivo and synergistically repress HIV expression from its LTR. In fission yeast, the RNA-induced transcriptional silencing (RITS) complex presents structural homology with HUSH. During transcription elongation by RNA polymerase II, RITS recruits a TRAMP-like RNA degradation complex composed of CNOT1 partners, MTR4 and the exosome, to ultimately repress gene expression via H3K9me3 deposit. Similarly, we show that TASOR interacts and cooperates with MTR4 and the exosome, in addition to CNOT1. We also highlight an interaction between TASOR and RNA Polymerase II, predominantly under its elongating state, and between TASOR and some HUSH-targeted nascent transcripts. Furthermore, we show that TASOR overexpression facilitates the association of the aforementioned RNA degradation proteins with RNA polymerase II. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral gene expression.

International audience; To evade host immune defences, human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2) have evolved auxiliary proteins that target cell restriction factors. Viral protein X (Vpx) from the HIV-2/SIVsmm lineage enhances viral infection by antagonizing SAMHD1 (refs 1,2), but this antagonism is not sufficient to explain all Vpx phenotypes. Here, through a proteomic screen, we identified another Vpx target-HUSH (TASOR, MPP8 and periphilin)-a complex involved in position-effect variegation3. HUSH downregulation by Vpx is observed in primary cells and HIV-2-infected cells. Vpx binds HUSH and induces its proteasomal degradation through the recruitment of the DCAF1 ubiquitin ligase adaptor, independently from SAMHD1 antagonism. As a consequence, Vpx is able to reactivate HIV latent proviruses, unlike Vpx mutants, which are unable to induce HUSH degradation. Although antagonism of human HUSH is not conserved among all lentiviral lineages including HIV-1, it is a feature of viral protein R (Vpr) from simian immunodeficiency viruses (SIVs) of African green monkeys and from the divergent SIV of l'Hoest's monkey, arguing in favour of an ancient lentiviral species-specific vpx/vpr gene function. Altogether, our results suggest the HUSH complex as a restriction factor, active in primary CD4+ T cells and counteracted by Vpx, therefore providing a molecular link between intrinsic immunity and epigenetic control.

Tat protein, the HIV transactivator, regulates transcription of the HIV genome by the host transcription machinery. Efficient inhibitors of HIV transcription that target Tat or the cellular cofactor NF-κB are well known. However, inhibition of HIV Tat-dependent transcription by targeting the general transcription and DNA repair factor II human (TFIIH) has not been reported. Here, we show that spironolactone (SP), an aldosterone antagonist approved for clinical use, inhibits HIV-1 and HIV-2 infection of permissive T cells by blocking viral Tat-dependent transcription from the long terminal repeat (LTR). We found that treatment of Jurkat and primary CD4 + T cells with SP induces degradation of the XPB cellular helicase, a component of the TFIIH complex, without affecting cellular mRNA levels, T cell viability, or T cell proliferation. We further demonstrate that the effect of SP on HIV infection is independent of its aldosterone antagonist function, since the structural analogue, eplerenone, does not induce XPB degradation and does not inhibit HIV infection. Rescue experiments showed that the SP-induced block of HIV infection relies, at least partially, on XPB degradation. In addition, we demonstrate that SP specifically inhibits Tat-dependent transcription, since basal transcription from the LTR is not affected. Our results demonstrate that SP is a specific inhibitor of HIV Tat-dependent transcription in T cells, which additionally suggests that XPB is a cofactor required for HIV infection. Targeting a cellular cofactor of HIV transcription constitutes an alternative strategy to inhibit HIV infection, together with the existing antiretroviral therapy. IMPORTANCE Transcription from the HIV promoter is regulated by the combined activities of the host transcription machinery and the viral transactivator Tat protein. Here, we report that the drug spironolactone—an antagonist of aldosterone—blocks viral Tat-dependent transcription, thereby inhibiting both HIV-1 and HIV-2 infection of permissive T cells. This inhibition relies on the degradation of the cellular helicase XPB, a component of the TFIIH transcription factor complex. Consequently, XPB appears to be a novel HIV cofactor. Our discovery of the HIV-inhibitory activity of spironolactone opens the way for the development of novel anti-HIV strategies targeting a cellular cofactor without the limitations of antiretroviral therapy of drug resistance and high cost.

International audience; Viruses often interfere with the DNA damage response to better replicate in their hosts. The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) protein has been reported to modulate the activity of the DNA repair structure-specific endonuclease subunit (SLX4) complex and to promote cell cycle arrest. Vpr also interferes with the base-excision repair pathway by antagonizing the uracil DNA glycosylase (Ung2) enzyme. Using an unbiased quantitative proteomic screen, we report that Vpr down-regulates helicase-like transcription factor (HLTF), a DNA translocase involved in the repair of damaged replication forks. Vpr subverts the DDB1-cullin4-associated-factor 1 (DCAF1) adaptor of the Cul4A ubiquitin ligase to trigger proteasomal degradation of HLTF. This event takes place rapidly after Vpr delivery to cells, before and independently of Vpr-mediated G2 arrest. HLTF is degraded in lymphocytic cells and macrophages infected with Vpr-expressing HIV-1. Our results reveal a previously unidentified strategy for HIV-1 to antagonize DNA repair in host cells.

Fast anterograde and retrograde axoplasmic transports in neurons rely on the activity of molecular motors and are critical for maintenance of neuronal and synaptic functions. Disturbances of axoplasmic transport have been identified in Alzheimer's disease and in animal models of this disease, but their mechanisms are not well understood. In this study we have investigated the distribution and the level of expression of kinesin light chains (KLCs) (responsible for binding of cargos during anterograde transport) and of dynein intermediate chain (DIC) (a component of the dynein complex during retrograde transport) in frontal cortex and cerebellar cortex of control subjects and Alzheimer's disease patients. By immunoblotting, we found a significant decrease in the levels of expression of KLC1 and 2 and DIC in the frontal cortex, but not in the cerebellar cortex, of Alzheimer's disease patients. A significant decrease in the levels of synaptophysin and of tubulin-β3 proteins, two neuronal markers, was also observed. KLC1 and DIC immunoreactivities did not co-localize with neurofibrillary tangles. The mean mRNA levels of KLC1, 2 and DIC were not significantly different between controls and AD patients. In SH-SY5Y neural cells, GSK-3β phosphorylated KLC1, a change associated to decreased association of KLC1 with its cargoes. Increased levels of active GSK-3β and of phosphorylated KLC1 were also observed in AD frontal cortex. We suggest that reduction of KLCs and DIC proteins in AD cortex results from both reduced expression and neuronal loss, and that these reductions and GSK-3β-mediated phosphorylation of KLC1 contribute to disturbances of axoplasmic flows and synaptic integrity in Alzheimer's disease., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

The complex bi-directional axoplasmic transport of mitochondria is essential for proper metabolic functioning of neurons and is controlled by phosphorylation. We have investigated by time-lapse imaging the effects of increased expression of glycogen synthase kinase-3beta (GSK-3beta) and of the p25 activator of cyclin dependent kinase 5 on mitochondria movements in mammalian cortical neurons and in PC12 cells. Both GSK-3beta and p25 increased the stationary behaviour of mitochondria in PC12 and in neurons, decreased their anterograde transport but did not affect the intrinsic velocities of mitochondria. The microtubule-associated tau proteins were more phosphorylated in GSK-3beta and p25 transfected neurons, but ultrastructural observation showed that these cells still contained microtubules and nocodazole treatment further reduced residual mitochondria movements in GSK-3beta or p25 transfected neurons, indicating that microtubule disruption was not the primary cause of increased mitochondrial stationary behaviour in GSK-3beta or p25 transfected neurons. Our results suggest that increased expression of GSK-3beta and p25 acted rather by decreasing the frequency of mitochondrial movements driven by molecular motors and that GSK-3beta and p25 might regulate these transports by controlling the time that mitochondria spend pausing, rather than their velocities.

Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the human immunodeficiency virus type 1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1(DCAF1) on Vpr stability. We show that the Vpr(Q65R) mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes wild-type Vpr and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr(Q65R) mutant. Conversely, small interfering RNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from human immunodeficiency virus type 2 and the SIVmac strain, results in increased G(2) arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1(DCAF1) complex. Furthermore, in human immunodeficiency virus type 1-infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1(DCAF1) acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counter-acting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest.

How the HIV1 Vpr protein initiates the host cell response leading to cell cycle arrest in G(2) has remained unknown. Here, we show that recruitment of DCAF1/VprBP by Vpr is essential for its cytostatic activity, which can be abolished either by single mutations of Vpr that impair DCAF1 binding, or by siRNA-mediated silencing of DCAF1. Furthermore, DCAF1 bridges Vpr to DDB1, a core subunit of Cul4 ubiquitin ligases. Altogether these results point to a mechanism where Vpr triggers G(2) arrest by hijacking the Cul4/DDB1(DCAF1) ubiquitin ligase. We further show that, Vpx, a non-cytostatic Vpr-related protein acquired by HIV2 and SIV, also binds DCAF1 through a conserved motif. Thus, Vpr from HIV1 and Vpx from SIV recruit DCAF1 with different physiological outcomes for the host cell. This in turn suggests that both proteins have evolved to preserve interaction with the same Cul4 ubiquitin ligase while diverging in the recognition of host substrates targeted for proteasomal degradation.

// Lauriane Velot 1,2,3,* , Angie Molina 1,2,3,* , Sylvie Rodrigues-Ferreira 1,2,3 , Anne Nehlig 1,2 , Benjamin Pierre Bouchet 4 , Marina Morel 3 , Ludovic Leconte 5 , Laurence Serre 6 , Isabelle Arnal 6 , Diane Braguer 7,8 , Ariel Savina 9 , Stephane Honore 7,8 and Clara Nahmias 1,2,3 1 Inserm U981, Institut Gustave Roussy Department of Molecular Medicine, Villejuif, France 2 Universite Paris-Saclay, Villejuif, France 3 CNRS UMR8104, Institut Cochin, Paris, France 4 Cell Biology, Faculty of Science, Utrecht University, Padualaan, CH Utrecht, The Netherlands 5 Cell and Tissue Imaging Core Facilty, PICT-IBiSA, CNRS UMR144 Institut Curie, Centre de Recherche, Paris, France 6 Inserm U836, Grenoble Institut des Neurosciences, Grenoble, France 7 Aix Marseille Universite, Inserm, CRO2 UMR_S 911, Marseille, France 8 APHM, Hopital Timone, Marseille, France 9 Scientific Partnerships Roche SAS, Boulogne Billancourt, France * These authors have contributed equally to this work Correspondence to: Clara Nahmias, email: // Keywords : EB1, MTUS1, protein interaction, +TIP, microtubule dynamics Received : May 07, 2015 Accepted : October 14, 2015 Published : October 20, 2015 Abstract The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.

Background SAMHD1 counteracts HIV-1 or HIV-2/SIVsmm that lacks Vpx by depleting the intracellular pool of nucleotides in myeloid cells and CD4+ quiescent T cells, thereby inhibiting the synthesis of retroviral DNA by reverse transcriptase. Depletion of nucleotides has been shown to underline the establishment of quiescence in certain cellular systems. These observations led us to investigate whether SAMHD1 could control the transition between proliferation and quiescence using the THP-1 cell model. Findings The entry of dividing THP-1 myeloid cells into a non-dividing differentiated state was monitored after addition of phorbol-12-myristate-13-acetate (PMA), an inducer of differentiation. Under PMA treatment, cells overexpressing SAMHD1 display stronger and faster adhesion to their support, compared to cells expressing a catalytically inactive form of SAMHD1, or cells depleted of SAMHD1, which appear less differentiated. After PMA removal, cells overexpressing SAMHD1 maintain low levels of cyclin A, in contrast to other cell lines. Interestingly, SAMHD1 overexpression slightly increases cell adhesion even in the absence of the differentiation inducer PMA. Finally, we found that levels of SAMHD1 are reduced in proliferating primary CD4+ T cells after T cell receptor activation, suggesting that SAMHD1 may also be involved in the transition from a quiescent state to a dividing state in primary T cells. Conclusions Altogether, we provide evidence that SAMHD1 may facilitate some aspects of THP-1 cell differentiation. Restriction of HIV-1 by SAMHD1 may rely upon its ability to modify cell cycle parameters, in addition to the direct inhibition of reverse transcription. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0425-y) contains supplementary material, which is available to authorized users.

Metastasis, a fatal complication of breast cancer, does not fully benefit from available therapies. In this study, we investigated whether ATIP3, the major product of 8p22 MTUS1 gene, may be a novel biomarker and therapeutic target for metastatic breast tumors. We show that ATIP3 is a prognostic marker for overall survival among patients with breast cancer. Notably, among metastatic tumors, low ATIP3 levels associate with decreased survival of the patients. By using a well-defined experimental mouse model of cancer metastasis, we show that ATIP3 expression delays the time-course of metastatic progression and limits the number and size of metastases in vivo. In functional studies, ATIP3 silencing increases breast cancer cell migration, whereas ATIP3 expression significantly reduces cell motility and directionality. We report here that ATIP3 is a potent microtubule-stabilizing protein whose depletion increases microtubule dynamics. Our data support the notion that by decreasing microtubule dynamics, ATIP3 controls the ability of microtubule tips to reach the cell cortex during migration, a mechanism that may account for reduced cancer cell motility and metastasis. Of interest, we identify a functional ATIP3 domain that associates with microtubules and recapitulates the effects of ATIP3 on microtubule dynamics, cell proliferation, and migration. Our study is a major step toward the development of new personalized treatments against metastatic breast tumors that have lost ATIP3 expression. Cancer Res; 73(9); 2905–15. ©2013 AACR.

Recent studies have highlighted the AT1 receptor as a potential therapeutic target in breast cancer, while the role of the AT2 subtype in this disease has remained largely neglected. The present study describes the generation and characterization of a new cellular model of human invasive breast cancer cells (D3H2LN-AT2) stably expressing high levels of Flag-tagged human AT2 receptor (Flag-hAT2). These cells exhibit high-affinity binding sites for AngII, and total binding can be displaced by the AT2-selective antagonist PD123319 but not by the AT1-selective antagonist losartan. Of interest, high levels of expression of luciferase and green fluorescent protein make these cells suitable for bioluminescence and fluorescence studies in vitro and in vivo. We provide here a novel tool to investigate the AT2 receptor functions in breast cancer cells, independently of AT1 receptor activation.

Human immunodeficiency virus type 2 (HIV-2), the second causative agent of human AIDS (5), arose from cross-transmission of sooty mangabey simian immunodeficiency virus (SIVsm), a lentivirus that naturally infects sooty mangabeys without inducing overt disease (14). Although AIDS caused by HIV-2 is as lethal as that caused by HIV-1, most HIV-2 carriers remain asymptomatic much longer than HIV-1 carriers (36). On the other hand, cross-transmission of SIVsm to rhesus macaques gave rise to highly pathogenic viral strains, and infected animals develop a disease similar to human AIDS (14). Thus, the SIVsm lentiviral lineage exhibits markedly different pathogenicities in its original and recently acquired hosts. It is currently unknown whether cross-species transmission of primate lentiviruses leads to specific functional changes in viral proteins that might directly modulate pathogenesis in the new host species. The HIV-1 and HIV-2 lentiviral lineages differ in the auxiliary proteins encoded by their genomes, which likely reflects different selective pressures in their adaptation to the host cell environment. Thus, the Vpx protein found in HIV-2 has no counterpart in HIV-1, whereas the genetically related Vpr protein is present in both lineages (47). Both Vpx and Vpr are actively packaged into the virions (1, 40), which suggests a role in early infection steps, i.e., prior to viral synthesis. Lack of Vpx strongly decreases the pathogenicity of SIVsmPBj in infected macaques (17). In vitro, Vpx is dispensable for infection of immortalized T cells but has been shown to be critical for primary macrophage infection (9, 23). In contrast, no cell culture system has so far revealed a strong requirement for Vpr in HIV infection, although Vpr-defective HIV-1 shows attenuated replication in human macrophages (2, 6, 16). Despite the absence of a clear defective phenotype of HIV lacking Vpr, this viral protein has so far attracted more attention than Vpx, presumably because of its intriguing ability to arrest the cell cycle at the G2/M transition (15, 21, 34, 35). We and others recently demonstrated that Vpr recruits DCAF1/VprBP, an adaptor of the CUL4A-DDB1 ubiquitin ligase complex (3, 8, 18, 26, 38, 46). A major role of ubiquitin ligases is the labeling of specific proteins for proteasome-mediated degradation, and the current hypothesis is that Vpr diverts the DCAF1 ubiquitin ligase to induce the degradation of a host protein required for entry into mitosis. We previously showed that Vpx from SIVsm also interacts with DCAF1, although unlike Vpr, it does not arrest the cell cycle (26). The ability to recruit DCAF1 might thus represent an ancient functional acquisition that predates the emergence of the genetically related but functionally distinct Vpr and Vpx genes. Here, we explore this hypothesis and address the potential role of Vpx-DCAF1 interaction in the context of HIV-2 replication in human macrophages.

HIV and SIV genomes encode several auxiliary proteins which have increasingly shown their importance in the virus-host relationship. One of these proteins, Vpx, is unique to the HIV-2/SIVsm lineage and is critical for viral replication in macrophages. The functional basis for this requirement as well as the Vpx mode of action have remained a mystery for quite some time. We have previously shown that HIV-1 Vpr induces cell cycle arrest by recruiting the CUL4A-DDB1DCAF1 ubiquitin ligase. This presumably leads to the degradation of a host factor required for cell cycle progression into mitosis. Vpr and Vpx proteins are evolutionary related and show significant sequence similarity even though they are not functionally redundant. This prompted us to address whether Vpx, like Vpr, recruits the CUL4A-DDB1DCAF1 ubiquitin ligase and whether Vpx uses this functional property to enable efficient macrophage infection by HIV-2. Confirming this hypothesis, our results show that DCAF1 is a critical host effector of Vpx in its ability to mediate infection and long-term replication of HIV-2 in human macrophages. WT Vpx associates with the DDB1 component of the CUL4A ubiquitin ligase through DCAF1 binding. In contrast the Q76R Vpx mutant is unable to bind DCAF1 and recruit DDB1. When placed in the HIV-2 provirus, Q76R Vpx severely diminished viral replication in macrophages. Vpx is incorporated into newly formed virions, suggesting an early function in the next infection cycle. Accordingly, precluding Vpx present in the incoming virions from recruiting DCAF1 in target macrophages leads to a postentry block characterized by defective accumulation of HIV-2 reverse transcripts. In addition Vpx from SIVsm functionally complements Vpx-defective HIV-2 in a DCAF1-binding dependent manner. Altogether, our data point to a mechanism in which Vpx diverts the DCAF1 ubiquitin ligase to inactivate an evolutionary conserved factor (RF: restriction factor), which restricts infection of macrophages by HIV-2 and closely related simian viruses (Figure 1). This function of Vpx challenges the previous idea that Vpx complements the lack of cellular factors necessary for viral replication in macrophages. Vpx therefore acts similarly to other HIV auxiliary proteins (Vif, Vpu, Nef) known to inactivate cellular factors in order to create an advantageous environment for the virus.

testing, a lithium chow diet was used. We also assessed the effect of the lithium diet on LTP of pyramidal cells in area CA1 of hippocampal slices, an area strongly implicated in the pathophysiology of Alzheimer’s disease. Socially housed rats were given continual access to lithium chow or to a control chow diet for four weeks prior to, and throughout all learning assessments. After four weeks access to their designated chow diet, rats began conditioning in the holeboard spatial discrimination or t-maze delayed alternation task. They immediately began conditioning in the opposite task once completing the first. This was then followed with social place-preference conditioning, after 24-hours isolation from their homecage social partner. Results: Results showed that chronic lithium magnified learning in all three paradigms, regardless of the reward received. Both food and social interaction supported enhanced learning. Furthermore, four weeks exposure to the lithium diet also enhanced LTP in area CA1 of hippocampal slices prepared from the rats fed with the lithium chow. This enhancement was shown under clinically relevant serum levels at the time of testing (0.70 0.03 mEq/l). Conclusions: These findings suggest that chronic lithium treatment may improve cognitive function in Alzheimer’s disease by enhancing mechanisms involved in basal learning and memory formation, such as hippocampal LTP.

Viral protein U (Vpu) of HIV-1 has two known functions in replication of the virus: degradation of its cellular receptor CD4 and enhancement of viral particle release. Vpu binds CD4 and simultaneously recruits the βTrCP subunit of the SCFβTrCP ubiquitin ligase complex through its constitutively phosphorylated DS52GXXS56 motif. In this process, Vpu was found to escape degradation, while inhibiting the degradation of βTrCP natural targets such as β-catenin and IκBα. We further addressed this Vpu inhibitory function with respect to the degradation of Emi1 and Cdc25A, two βTrCP substrates involved in cell-cycle progression. In the course of these experiments, we underscored the importance of a novel phosphorylation site in Vpu. We show that, especially in cells arrested in early mitosis, Vpu undergoes phosphorylation of the serine 61 residue, which lies adjacent to the βTrCP-binding motif. This phosphorylation event triggers Vpu degradation by a βTrCP-independent process. Mutation of Vpu S61 in the HIV-1 provirus extends the half-life of the protein and significantly increases the release of HIV-1 particles from HeLa cells. However, the S61 determinant of regulated Vpu turnover is highly conserved within HIV-1 isolates. Altogether, our results highlight a mechanism where differential phosphorylation of Vpu determines its fate as an adaptor or as a substrate of distinct ubiquitin ligases. Conservation of the Vpu degradation determinant, despite its negative effect on virion release, argues for a role in overall HIV-1 fitness., Author Summary In addition to the structural and catalytic proteins characteristic of retroviruses, HIV-1 encodes auxiliary proteins that govern virus-host interaction. One such auxiliary protein termed Vpu both enhances virion release from human cells and the degradation of CD4, the cellular surface receptor of HIV-1. The latter activity relies on a strategy frequently used by pathogens, i.e., the hijacking of the protein degradation machinery. This process involves the ubiquitin ligases that ensure the selection of the protein subsequently degraded. Vpu interacts both with an ubiquitin ligase and CD4, bridging the latter to the degradation machinery. Here, we show that in addition to subverting one particular ubiquitin ligase to trigger CD4 degradation, Vpu is itself recognized in a phosphorylation-dependent manner by a distinct ubiquitin ligase and degraded. Mutations of Vpu that confer resistance to this degradation process enhance viral particle release, whereas the determinant of Vpu turnover is paradoxically well conserved among HIV-1 isolates. These results suggest that the virus is constrained to limit its own production for long-term persistence.

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.