RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of HIV-1

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential to mediate oligomerization of the protein. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function

Etude des kinases assurant l'activation cellulaire des inhibiteurs nucléosidiques utilisés dans les traitements antiviraux

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Retrovolution: HIV-driven evolution of cellular genes and improvement of anticancer drug activation.

In evolution strategies aimed at isolating molecules with new functions, screening for the desired phenotype is generally performed in vitro or in bacteria. When the final goal of the strategy is the modification of the human cell, the mutants selected with these preliminary screenings may fail to confer the desired phenotype, due to the complex networks that regulate gene expression in higher eukaryotes. We developed a system where, by mimicking successive infection cycles with HIV-1 derived vectors containing the gene target of the evolution in their genome, libraries of gene mutants are generated in the human cell, where they can be directly screened. As a proof of concept we created a library of mutants of the human deoxycytidine kinase (dCK) gene, involved in the activation of nucleoside analogues used in cancer treatment, with the aim of isolating a variant sensitizing cancer cells to the chemotherapy compound Gemcitabine, to be used in gene therapy for anti-cancer approaches or as a poorly immunogenic negative selection marker for cell transplantation approaches. We describe the isolation of a dCK mutant, G12, inducing a 300-fold sensitization to Gemcitabine in cells originally resistant to the prodrug (Messa 10K), an effect 60 times stronger than the one induced by the wt enzyme. The phenotype is observed in different tumour cell lines irrespective of the insertion site of the transgene and is due to a change in specificity of the mutated kinase in favour of the nucleoside analogue. The mutations characterizing G12 are distant from the active site of the enzyme and are unpredictable on a rational basis, fully validating the pragmatic approach followed. Besides the potential interest of the G12 dCK variant for therapeutic purposes, the methodology developed is of interest for a large panel of applications in biotechnology and basic research

Degradation-Independent Inhibition of APOBEC3G by the HIV-1 Vif Protein

The ubiquitin–proteasome system plays an important role in the cell under normal physiological conditions but also during viral infections. Indeed, many auxiliary proteins from the (HIV-1) divert this system to its own advantage, notably to induce the degradation of cellular restriction factors. For instance, the HIV-1 viral infectivity factor (Vif) has been shown to specifically counteract several cellular deaminases belonging to the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 or A3) family (A3A to A3H) by recruiting an E3-ubiquitin ligase complex and inducing their polyubiquitination and degradation through the proteasome. Although this pathway has been extensively characterized so far, Vif has also been shown to impede A3s through degradation-independent processes, but research on this matter remains limited. In this review, we describe our current knowledge regarding the degradation-independent inhibition of A3s, and A3G in particular, by the HIV-1 Vif protein, the molecular mechanisms involved, and highlight important properties of this small viral protein

YTHDC1 regulates distinct post-integration steps of HIV-1 replication and is important for viral infectivity

International audienceBackground The recent discovery of the role of m 6 A methylation in the regulation of HIV-1 replication unveiled a novel layer of regulation for HIV gene expression. This epitranscriptomic modification of HIV-1 RNAs is under the dynamic control of specific writers and erasers. In addition, cytoplasmic readers of the m 6 A mark are recruited to the modified viral RNAs and regulate HIV-1 replication. Yet, little is known about the effects of m 6 A writers and readers on the biogenesis of HIV-1 RNAs. Results We showed that the METTL3/14 m 6 A methyltransferase complex and the m 6 A YTHDF2 cytoplasmic writer down regulates the abundance of HIV-1 RNAs in infected cells. We also identified the m 6 A nuclear writer YTHDC1 as a novel regulator of HIV-1 transcripts. In HIV-1 producer cells, we showed that knocking down YTHDC1 increases the levels of unspliced and incompletely spliced HIV-1 RNAs, while levels of multiply spliced transcripts remained unaffected. In addition, we observed that depletion of YTHDC1 has no effect on the nuclear cytoplasmic distribution of viral transcripts. YTHDC1 binds specifically to HIV-1 transcripts in a METTL3-dependent manner. Knocking down YTHDC1 reduces the expression of Env and Vpu viral proteins in producer cells and leads to the incorporation of unprocessed Env gp160 in virus particles, resulting in the decrease of their infectivity. Conclusions Our findings indicate that, by controlling HIV-1 RNA biogenesis and protein expression, the m 6 A nuclear reader YTHDC1 is required for efficient production of infectious viral particles. Graphical Abstrac

Identification of HIV-1 sequences targeted by the miRNA Induced silencing complex (miRISC) using Argonaute 2 cross-linking and immunoprecipitation (HITS-CLIP)

International audienc

Antiviral Protein APOBEC3G Localizes to Ribonucleoprotein Complexes Found in P Bodies and Stress Granules

Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function

The system generates genetic diversity.

<p><i>Panel A</i>. Expected relationship between the size of the target gene and the number of Retrovolution cycles required to obtain a library containing one mutated position per virion in the gene sequence, based on the mutation rate of 3.4×10⁻⁵ mutations per nt per cycle, described in the literature <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002904#pgen.1002904-Mansky2" target="_blank">[3]</a> (dashed line), and based on the rate observed experimentally in this work (solid black line). <i>Panel B</i>. Distribution of the mutated positions within the dCK coding sequence (783 bp) found by sequencing 40 clones at generation 16. <i>Panel C</i>. Position of the 12 mutations detected in the U3 region of the LTRs of all vectors sequenced starting from generation 8 (included).</p

Screening of the dCK library.

<p><i>Panel A</i>. Viability of 76 isolated clones of HEK-293T transduced with the dCK-F16 viral library at a MOI<1 in the presence of 10 nM Gemcitabine. The different colors represent the percentage of dead cells on the total of seeded cells. Each line corresponds to an independent experiment, and each column to one clone. The results obtained with untransduced cells and cells transduced with the wt-dCK gene are given on the right (“HEK-293T” and “wt-dCK”, columns, respectively). <i>Panel B</i>. Viability of selected Messa10K “polyclonal populations” in the presence of increasing concentrations of Gemcitabine. Thick pale blue line, untransduced Messa10K cells; thick red line, cells transduced with wt-dCK; thin lines, cells transduced with the vectors derived from the clones identified in HEK-293T (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002904#pgen-1002904-g001" target="_blank">Figure 1D</a>): pink, B9; gray, B5; dark blue, F8; purple, G7; orange, E8; green, G12. Values represent the ratio of the number of live cells over the total of cells seeded for each Gemcitabine concentration. For each dCK variant, 4 to 9 populations of Messa10K cells were generated independently, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002904#pgen.1002904.s001" target="_blank">Figure S1</a>, and for each population the experiment was carried out in duplicate. For the sake of clarity, the error bars are shown only for samples G12 and wt-dCK. The values of G12 and wt-dCK are significantly different for every Gemcitabine concentration tested (p<0.0001). <i>Panel C</i>. Cell death for Messa10K “polyclonal populations” either non-transduced (light blue) or transduced with the wt-dCK (red) or the G12 variant (green), with a larger range of Gemcitabine concentrations than in panel B. The arrows indicate the IC50 values for Gemcitabine in the three different cell types.</p

The sensitization phenotype is due to a modified dCK activity.

<p><i>Panel A</i>. Western Blot analysis of dCK expression levels in different “polyclonal populations”. M: untransduced Messa10K cells; M-dCK: 4 Messa10K populations transduced with wt-dCK gene; M-G12: 4 Messa10K populations transduced with the G12 variant. For each population 6,12 and 24 µg of total protein were loaded on the gel. <i>Panel B</i>. Gemcitabine sensitivity of Messa10K cells transduced with vectors bearing the G12 mutant sequence inserted in a wild-type backbone. Triangles: untransduced cells. Squares: cells transduced with wt-dCK. Empty circles: cells transduced with G12. Full circles: cells transduced with G12/wt-backbone. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002904#s2" target="_blank">Results</a> are the average of 6 independent experiments. <i>Panels C and D</i>. Sensitivity to Gemcitabine of HT29 cells (panel C) and of BxPC3 cells (panel D) transduced with one copy of G12 variant per cell. Triangles: untransduced cells. Squares: cells transduced with a wt-dCK vector. Circles: cells transduced with a G12 vector. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002904#s2" target="_blank">Results</a> are the average of 2 independent experiments.</p