DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence.

BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. RESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. CONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.

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.

Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This "mito-aggresome" is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

The viral restriction factor tetherin/BST2 tethers cytokinetic midbody remnants to the cell surface.

The midbody at the center of the intercellular bridge connecting dividing cells recruits the machinery essential for the final steps of cytokinesis.1-5 Successive abscission on both sides of the midbody generates a free midbody remnant (MBR) that can be inherited and accumulated in many cancer, immortalized, and stem cells, both in culture and in vivo.6-12 Strikingly, this organelle was recently shown to contain information that induces cancer cell proliferation, influences cell polarity, and promotes dorso-ventral axis specification upon interaction with recipient cells.13-16 Yet the mechanisms by which the MBR is captured by either a daughter cell or a distant cell are poorly described.10,14 Here, we report that BST2/tetherin, a well-established restriction factor that blocks the release of numerous enveloped viruses from the surface of infected cells,17-20 plays an analogous role in retaining midbody remnants. We found that BST2 is enriched at the midbody during cytokinesis and localizes at the surface of MBRs in a variety of cells. Knocking out BST2 induces the detachment of MBRs from the cell surface, their accumulation in the extracellular medium, and their transfer to distant cells. Mechanistically, the localization of BST2 at the MBR membrane is both necessary and sufficient for the interaction between MBRs and the cell surface. We thus propose that BST2 tethers post-cytokinetic midbody remnants to the cell surface. This finding reveals new parallels between cytokinesis and viral biology21-26 that unexpectedly extend beyond the ESCRT-dependent abscission step.

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.

TIM-1 Ubiquitination Mediates Dengue Virus Entry.

Dengue virus (DENV) is a major human pathogen causing millions of infections yearly. Despite intensive investigations, a DENV receptor that directly participates in virus internalization has not yet been characterized. Here, we report that the phosphatidylserine receptor TIM-1 is an authentic DENV entry receptor that plays an active role in virus endocytosis. Genetic ablation of TIM-1 strongly impaired DENV infection. Total internal reflection fluorescence microscopy analyses of live infected cells show that TIM-1 is mostly confined in clathrin-coated pits and is co-internalized with DENV during viral entry. TIM-1 is ubiquitinated at two lysine residues of its cytoplasmic domain, and this modification is required for DENV endocytosis. Furthermore, STAM-1, a component of the ESCRT-0 complex involved in intracellular trafficking of ubiquitinated cargos, interacts with TIM-1 and is required for DENV infection. Overall, our results show that TIM-1 is the first bona fide receptor identified for DENV.

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.

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.

Endosomal trafficking of HIV-1 gag and genomic RNAs regulates viral egress.

HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVamp-reduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.

Contribution of endocytic motifs in the cytoplasmic tail of herpes simplex virus type 1 glycoprotein B to virus replication and cell-cell fusion.

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.

The clathrin adaptor complex AP-1 binds HIV-1 and MLV Gag and facilitates their budding.

Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1mu obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1mu enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1mu. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners.

Luman, a new partner of HIV-1 TMgp41, interferes with Tat-mediated transcription of the HIV-1 LTR.

In our search for new partners of the HIV-1 envelope glycoprotein (Env), we found that the cytoplasmic domain of the TMgp41 (TMgp41 CD) subunit of HIV-1 Env interacted with Luman, a transcription factor of the CREB/ATF family. Luman is anchored in the endoplasmic reticulum membrane and subjected to activation by regulated intramembrane proteolysis (RIP). The RIP process permits the release of the activated amino-terminal fragment of Luman into the cytoplasm, and its import into the nucleus. Here, we demonstrate that interaction between the TMgp41 CD and Luman requires a region encompassing the b-Zip and TM domains of Luman and decreases the stability of this factor. Moreover, we found that overexpression of a constitutively active form of Luman in cells transfected with HXB2R HIV-1 provirus decreased the intracellular expression of Gag and Env and led to a decrease in virion release. This negative effect of activated Luman on HIV-1 production was correlated to the inhibition of Tat transactivation of the HIV-1 LTR, which might be related to an interaction of activated Luman with Tat. Altogether, these results show that Luman acts as a partner of two major HIV-1 proteins: the TMgp41 Env subunit and Tat. The interaction between the TMgp41 subunit of Env and Luman affects the stability of the full-length Luman protein, the precursor of the activated, nuclear form of Luman, which acts negatively on Tat-mediated HIV-1 transactivation.

The conserved dileucine- and tyrosine-based motifs in MLV and MPMV envelope glycoproteins are both important to regulate a common Env intracellular trafficking.

BACKGROUND: Retrovirus particles emerge from the assembly of two structural protein components, Gag that is translated as a soluble protein in the cytoplasm of the host cells, and Env, a type I transmembrane protein. Because both components are translated in different intracellular compartments, elucidating the mechanisms of retrovirus assembly thus requires the study of their intracellular trafficking. RESULTS: We used a CD25 (Tac) chimera-based approach to study the trafficking of Moloney murine leukemia virus and Mason-Pfizer monkey virus Env proteins. We found that the cytoplasmic tails (CTs) of both Env conserved two major signals that control a complex intracellular trafficking. A dileucine-based motif controls the sorting of the chimeras from the trans-Golgi network (TGN) toward endosomal compartments. Env proteins then follow a retrograde transport to the TGN due to the action of a tyrosine-based motif. Mutation of either motif induces the mis-localization of the chimeric proteins and both motifs are found to mediate interactions of the viral CTs with clathrin adaptors. CONCLUSION: This data reveals the unexpected complexity of the intracellular trafficking of retrovirus Env proteins that cycle between the TGN and endosomes. Given that Gag proteins hijack endosomal host proteins, our work suggests that the endosomal pathway may be used by retroviruses to ensure proper encountering of viral structural Gag and Env proteins in cells, an essential step of virus assembly.

Tail-interacting protein TIP47 is a connector between Gag and Env and is required for Env incorporation into HIV-1 virions.

The presence of the envelope glycoprotein Env in HIV-1 virions is essential for infectivity. To date, the molecular mechanism by which Env is packaged into virions has been largely unknown. Here, we show that TIP47 (tail-interacting protein of 47 kDa), which has been shown to interact with Env, also binds the MA (matrix) domain of HIV-1 Gag protein and that these three proteins form a ternary complex. Mutations in Gag that abrogate interaction with TIP47 inhibit Env incorporation and virion infectivity as well as colocalization between Gag and Env. We also show that TIP47 silencing impairs Env incorporation and infectivity and abolishes coimmunoprecipitation of Gag with Env. In contrast, overexpression of TIP47 increases Env packaging. Last, we demonstrate that TIP47 can interact simultaneously with Env and Gag. Taken together, our results show that TIP47 is a cellular cofactor that plays an essential role in Env incorporation, allowing the encounter and the physical association between HIV-1 Gag and Env proteins during the viral assembly process.