Lentiviral vectors and antiretroviral intrinsic immunity

Multicellular organisms have evolved under relentless attacks from pathogens, and as a consequence have spiked their genomes with numerous genes that serve to thwart these threats, notably through the building of the innate and adaptive arms of the immune system. The innate immune system is by far the most ancient, being found as widely as in plants and Drosophila, while adaptive immunity arose with the emergence of cartilaginous fishes. Innate immunity enters rapidly into the game during the course of an infection and generally involves the recognition by specific cellular receptors of common pathogen-associated patterns to elicit broad defensive responses, mediated in humans by interferons, macrophages, and natural killer cells, amongst others. When innate immunity fails to eradicate the infection quickly, adaptive immune responses enter into play, to generate exquisitely specific defenses to virtually any pathogen, thanks to a quasi-infinite repertoire of nonself receptors and effectors. A specific form of innate immunity, coined "intrinsic immunity," completes this protection by providing a constant, always-on, line of defense, generally through intracellular obstacles to the replication of pathogens. This component of the immune system has gained much attention as it was discovered that it is a cornerstone of the resistance of mammals against retroviruses. One of these newly discovered intracellular molecular weapons, the APOBEC family of proteins, is active against several classes of retroelements. We present here the current state of knowledge on this rapidly evolving field and discuss implications for gene therapy

Characterization of APOBEC3G binding to 7SL RNA

Human APOBEC3 proteins are editing enzymes that can interfere with the replication of exogenous retroviruses such as human immunodeficiency virus (HIV), hepadnaviruses such as hepatitis B virus (HBV), and with the retrotransposition of endogenous retroelements such as long-interspersed nuclear elements (LINE) and Alu. Here, we show that APOBEC3G, but not other APOBEC3 family members, binds 7SL RNA, the common ancestor of Alu RNAs that is specifically recruited into HIV virions. Our data further indicate that APOBEC3G recognizes 7SL RNA and Alu RNA by its common structure, the Alu domain, suggesting a mechanism for APOBEC3G- mediated inhibition of Alu retrotransposition. However, we also demonstrate that APOBEC3F and APOBEC3G are normally recruited into and inhibit the infectivity of ΔVif HIV1 virions when 7SLRNA is prevented from accessing particles by RNA interference against SRP14 or by over expression of SRP19, both components of the signal recognition particle. We thus conclude that 7SL RNA is not an essential mediator of the virion packaging of these antiviral cytidine deaminases

Inhibition of HIV-1 replication by balsamin, a ribosome inactivating protein of Momordica balsamina

Ribosome-inactivating proteins (RIPs) are endowed with several medicinal properties, including antiviral activity. We demonstrate here that the recently identified type I RIP from Momordica balsamina also possesses antiviral activity, as determined by viral growth curve assays and single-round infection experiments. Importantly, this activity is at play even as doses where the RIP has no cytotoxic effect. In addition, balsamin inhibits HIV-1 replication not only in T cell lines but also in human primary CD4(+) T cells. This antiviral compound exerts its activity at a viral replicative step occurring later than reverse-transcription, most likely on viral protein translation, prior to viral budding and release. Finally, we demonstrate that balsamin antiviral activity is broad since it also impedes influenza virus replication. Altogether our results demonstrate that type I RIP can exert a potent anti-HIV-1 activity which paves the way for new therapeutic avenues for the treatment of viral infections

APOBEC3G/3F mediates intrinsic resistance of monocyte-derived dendritic cells to HIV-1 infection

HIV-1 infects immature dendritic cells (iDCs), but infection is inefficient compared with activated CD4+ T cells and only involves a small subset of iDCs. We analyzed whether this could be attributed to specific cellular restrictions during the viral life cycle. To study env-independent restriction to HIV-1 infection, we used a single-round infection assay with HIV-1 pseudotyped with vesicular stomatitis virus G protein (HIV-VSVG). Small interfering RNA–mediated depletion of APOBEC3G/3F (A3G/3F), but not TRIM5α, enhanced HIV-1 infection of iDCs, indicating that A3G/3F controls the sensitivity of iDCs to HIV-1 infection. Furthermore, sequences of HIV reverse transcripts revealed G-to-A hypermutation of HIV genomes during iDC infection, demonstrating A3G/3F cytidine deaminase activity in iDCs. When we separated the fraction of iDCs that was susceptible to HIV, we found the cells to be deficient in A3G messenger RNA and protein. We also noted that during DC maturation, which further reduces susceptibility to infection, A3G levels increased. These findings highlight a role for A3G/3F in explaining the resistance of most DCs to HIV-1 infection, as well as the susceptibility of a fraction of iDCs. An increase in the A3G/3F-mediated intrinsic resistance of iDCs could result in a block of HIV infection at its mucosal point of entry

Model Structure of Human APOBEC3G

BACKGROUND: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance. METHODOLOGY/PRINCIPAL FINDINGS: We predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering ΔVif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface. CONCLUSIONS/SIGNIFICANCE: The structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G

Quantitative Multicolor Super-Resolution Microscopy Reveals Tetherin HIV-1 Interaction

Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resolution analysis of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, respectively. Multicolor super-resolution microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters containing on average 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochemical and super-resolution analysis revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resolution microscopy analysis of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions

HIV-1 Vpu Neutralizes the Antiviral Factor Tetherin/BST-2 by Binding It and Directing Its Beta-TrCP2-Dependent Degradation

Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and β-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a β-TrCP2-dependent manner. Accordingly, in conditions where Vpu-β-TrCP2-tetherin interplay was not operative, including cells stably knocked down for β-TrCP2 expression or cells expressing a dominant negative form of β-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its β-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication

La tyrosine kinase Syk influence négativement la progression du cycle cellulaire à travers la phosphorylation de la kinase Cdk1

La tyrosine kinase cytoplasmique Syk a longtemps été étudiée dans les cellules hématopoïétiques en tant qu'acteur de la réponse immunitaire. Notre laboratoire a montré, pour la première fois, une expression de Syk dans les cellules et tissus mammaires humains sains et faiblement tumorigéniques, alors qu'elle est absente ou peu exprimée dans les cellules et carcinomes mammaires invasifs. La transfection de Syk dans une lignée de cancer du sein fortement tumorigénique, abolit sa capacité tumorale et métastastique chez la souris athymique, conférant à Syk un rôle de suppresseur de tumeurs. Curieusement, la surexpression de Syk par transfection provoque des défauts de division cellulaire, et induit une mort cellulaire, évoquant la catastrophe mitotique. De plus notre laboratoire a montré que Syk est localisée et catalytiquement active au niveau des centrosomes, et que son expression y est régulée de façon spatio-temporelle au cours du cycle cellulaire. Néanmoins, les mécanismes moléculaires ainsi que les effecteurs de Syk responsables de son activité anti-oncogénique demeurent inconnus. Dans cette thèse nous montrons que Syk pourrait agir de façon négative sur le déroulement de la division cellulaire à travers la phosphorylation d'une protéine clef de la mitose: la kinase Cdk1. Nous avons observé que Syk interagit avec le complexe Cdk1/cycline B1 et phosphoryle Cdk1 sur différents résidus tyrosine aux centrosomes dont la tyrosine 15, connue pour influencer négativement le déroulement de la mitose. Etonnamment, la surexpression transitoire de Syk provoque une accumulation des cellules en phase G1 du cycle cellulaire. Enfin, nous démontrons que des agents anticancéreux provoquant du stress génotoxique et bloquant le cycle cellulaire, induisent la phosphorylation de Cdk1 sur tyrosine dépendant de l'activation de Syk. Ces fonctions nouvelles de Syk contribueront à une meilleure compréhension de son activité anti-oncogénique dans les cellules de cancer du seinThe non-receptor Syk tyrosine kinase has mainly been studied in haematopoietic cells in which it plays a key role in the immune-response signalling. Our laboratory demonstrated for the first time that Syk is expressed also in normal human breast cells and tissue and low-tumorigenic breast cancer cell lines, whereas its expression is low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of Syk in a highly tumorigenic breast cancer cell line suppressed its tumorigenic and metastatic capacity in athymic mice, suggesting that Syk acts as a tumour suppressor. Surprisingly, overexpression of transfected Syk provokes abnormal cell division and a non-apoptotic cell death, reminiscent of mitotic catastrophe. Furthermore, our laboratory demonstrated that Syk is localized and catalytically active at the centrosomes, in which Syk expression is controlled in a spatio-temporal manner. Nevertheless, the molecular mechanisms and the Syk effectors responsible for its anti-oncogenic activity remain unknown. In this PhD thesis, we demonstrate that Syk could negatively affect cell division through the phosphorylation of a key protein involved in the control of mitosis: the Cdk1 kinase. We observed that Syk interacts with the Cdk1/cyclin B1 complex and that it phosphorylates Cdk1 on different tyrosine residues, amongst which we identified the tyrosine 15 residue, known to negatively affect the progression of mitosis. Surprisingly, transient Syk overexpression induced an accumulation of cells in the G1 cell cycle phase. Finally, we demonstrate that anti-cancer drugs that provoke genotoxic stress and a cell cycle block induce phosphorylation of Cdk1 on tyrosine and this in a Syk activation-dependent manner. These novel aspects of Syk function will undeniably contribute to a better understanding of its onco-suppressive activity in breast cancer cellsMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF