From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

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Regulation of Viral Restriction by Post-Translational Modifications

Intrinsic immunity is orchestrated by a wide range of host cellular proteins called restriction factors. They have the capacity to interfere with viral replication, and most of them are tightly regulated by interferons (IFNs). In addition, their regulation through post-translational modifications (PTMs) constitutes a major mechanism to shape their action positively or negatively. Following viral infection, restriction factor modification can be decisive. Palmitoylation of IFITM3, SUMOylation of MxA, SAMHD1 and TRIM5α or glycosylation of BST2 are some of those PTMs required for their antiviral activity. Nonetheless, for their benefit and by manipulating the PTMs machinery, viruses have evolved sophisticated mechanisms to counteract restriction factors. Indeed, many viral proteins evade restriction activity by inducing their ubiquitination and subsequent degradation. Studies on PTMs and their substrates are essential for the understanding of the antiviral defense mechanisms and provide a global vision of all possible regulations of the immune response at a given time and under specific infection conditions. Our aim was to provide an overview of current knowledge regarding the role of PTMs on restriction factors with an emphasis on their impact on viral replication

MoMuLV and HIV-1 Nucleocapsid Proteins Have a Common Role in Genomic RNA Packaging but Different in Late Reverse Transcription

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Internal RNA 2′-O-methylations on HIV-1 genome impair reverse transcription.

Viral RNA genomes are modified by epitranscriptomic marks, including 2′-O-methylations that are added by cellular or viral methyltransferases. 2′-O-methylations modulate RNA structure, function, and discrimination between self and non-self RNA by innate immune sensors such as RIG-I like receptors. This is illustrated by HIV-1 that decorates its RNA genome through hijacking the cellular FTSJ3 2′-O-methyltransferase, thereby limiting immune sensing and interferon production. However, the impact of such RNA modification during viral genome replication is poorly understood. Here we show by performing endogenous reverse transcription on methylated or hypomethylated HIV-1 particles, that 2′-O-methylations negatively affect HIV-1 reverse transcriptase (RT) activity. Biochemical assays confirm that RNA 2′-O-methylation impedes RT activity, especially at low dNTP concentrations reflecting those in quiescent cells, by reducing nucleotide incorporation efficiency and impairing translocation. Mutagenesis highlights K70 as a critical amino-acid for the RT to bypass 2′-O-methylations. Hence, the observed antiviral effect due to viral RNA 2′-O-methylation antagonizes the FTSJ3-mediated proviral effects, suggesting the fine tuning of RNA methylation during viral replication.Les génomes d'ARN viraux sont modifiés par des marques épitranscriptomiques, notamment des 2′-O-méthylations ajoutées par des méthyltransférases cellulaires ou virales. Les 2′-O-méthylations modulent la structure et la fonction de l'ARN, ainsi que la discrimination entre l'ARN du soi et celui du non-soi par des capteurs immunitaires innés tels que les récepteurs de type RIG-I. Ceci est illustré par le VIH-1 qui décore son génome ARN en détournant la 2′-O-méthyltransférase cellulaire FTSJ3, limitant ainsi la détection immunitaire et la production d'interféron. Cependant, l'impact d'une telle modification de l'ARN pendant la réplication du génome viral est mal compris. Nous montrons ici, en effectuant une transcription inverse endogène sur des particules de VIH-1 méthylées ou hypométhylées, que les 2′-O-méthylations affectent négativement l'activité de la transcriptase inverse (TI) du VIH-1. Les essais biochimiques confirment que la 2′-O-méthylation de l'ARN entrave l'activité de la transcriptase inverse, en particulier à de faibles concentrations de dNTP reflétant celles des cellules quiescentes, en réduisant l'efficacité de l'incorporation des nucléotides et en entravant la translocation. La mutagenèse met en évidence le fait que K70 est un acide aminé critique permettant à la RT de contourner les 2′-O-méthylations. Par conséquent, l'effet antiviral observé dû à la 2′-O-méthylation de l'ARN viral antagonise les effets proviraux médiés par FTSJ3, ce qui suggère un réglage fin de la méthylation de l'ARN au cours de la réplication virale

Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly

International audienceHIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT

Internal RNA 2′- O -methylation on the HIV-1 genome impairs reverse transcription

International audienceAbstract Viral RNA genomes are modified by epitranscriptomic marks, including 2′-O-methylation that is added by cellular or viral methyltransferases. 2′-O-Methylation modulates RNA structure, function and discrimination between self- and non-self-RNA by innate immune sensors such as RIG-I-like receptors. This is illustrated by human immunodeficiency virus type-1 (HIV-1) that decorates its RNA genome through hijacking the cellular FTSJ3 2′-O-methyltransferase, thereby limiting immune sensing and interferon production. However, the impact of such an RNA modification during viral genome replication is poorly understood. Here we show by performing endogenous reverse transcription on methylated or hypomethylated HIV-1 particles, that 2′-O-methylation negatively affects HIV-1 reverse transcriptase activity. Biochemical assays confirm that RNA 2′-O-methylation impedes reverse transcriptase activity, especially at low dNTP concentrations reflecting those in quiescent cells, by reducing nucleotide incorporation efficiency and impairing translocation. Mutagenesis highlights K70 as a critical amino acid for the reverse transcriptase to bypass 2′-O-methylation. Hence, the observed antiviral effect due to viral RNA 2′-O-methylation antagonizes the FTSJ3-mediated proviral effects, suggesting the fine-tuning of RNA methylation during viral replication

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community

Quantitative analysis of the nucleic acid content of viral particles released from MuLV producer cells.

<p>(A) Quantitation of viral gRNA incorporated in wt or mutant viruses by RT-QPCR. Mock controls were subtracted from assays. Error bars indicate SD from at least four independent experiments. (B) Viral DNA levels were determined by qPCR in the wt and mutant virions. DNA was extracted from same virion samples as those used before for gRNA quantitation. Error bars indicate SD from at least seven independent experiments. (C) There is no correlation between gRNA and viral DNA levels among the MuLV mutants. For comparative purpose, data obtained with HIV-1 virions deleted of the second ZF (ΔZF2) are given (left part) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051534#pone.0051534-Houzet1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051534#pone.0051534-Meric1" target="_blank">[38]</a>. To facilitate the comparison, levels of viral gRNA and ss-cDNA were normalized to those measured in wt virions.</p

Strategy of qPCR to monitor the MuLV nucleic acid species.

<p>Templates and primers used for qPCR analyses were schematically represented. Only the spliced SD' RNA (SD'/SA) important for this study and the gRNA are indicated. A color code was used to illustrate the specificity of the PCR-primer pairs (arrows) that were used to quantify the pR88 plasmid (blue) which generates the MuLV gRNA transcript (orange) and the spliced SD' RNA (green). Numbers refer to the position of the elongation start. Bottom panel: Products of viral reverse transcription. The primer pairs used to detect the intermediate ss-cDNA (red), Pol cDNA (orange), SD' cDNA (green) and the final product FL DNA (purple) are shown.</p

Imaging HIV-1 RNA dimerization in cells by multicolor super-resolution and fluctuation microscopies

International audienceDimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process