Caractérisation fonctionnelle de la protéine précoce-immédiate 2 de l'herpèsvirus humain 6

L’herpèsvirus humain 6 (HHV-6) est un pathogène opportuniste dont l’infection et/ou la réactivation sont associées avec des maladies telles la roséole, des désordres du système nerveux central, et des complications suite à la transplantation d’organes. Le séquençage du génome viral a révélé l’existence de deux types de HHV-6 (A et B), se distinguant par des séquences dissemblables dans des régions spécifiques et des propriétés biologiques différentes. Nos travaux portent sur la caractérisation de la protéine précoce-immédiate (IE) 2 de HHV-6A. Son expression précoce suite à l’infection et sa capacité de transactivation suggèrent qu’elle représente une protéine clé dans l’établissement d’une infection productive, grâce à son contrôle de la cascade d’expression des gènes viraux. De plus, le transcrit codant pour IE2 se situe dans la région la plus variable entre HHV-6A et -6B, suggérant que la biologie de cette protéine pourrait contribuer à expliquer les différences cliniques entre les deux types viraux. Afin de déterminer les protéines cellulaires recrutées par IE2 durant l’établissement de l’infection, nous avons criblé une librairie de cellules T à la recherche de partenaires d’interaction. Nous avons isolé la protéine Ubc9, enzyme jouant un rôle dans la voie de conjugaison de SUMO. Cette interaction a une importance fonctionnelle pour IE2, puisqu’Ubc9 réprime significativement l’activation de promoteurs par la protéine virale. Les domaines essentiels à la fonctionnalité d’IE2 n’avaient jamais été caractérisés. Nous avons démontré que les domaines N- et C-terminaux sont tous deux requis pour une transactivation optimale, et que la délétion de la queue C-terminale d’IE2 modifie significativement la transactivation et la localisation intranucléaire de la protéine. Nous avons également établi que la région R3 du promoteur précoce-immédiat de HHV-6A est positivement régulée par IE2. Globalement, ces travaux nous procurent une vision plus précise du rôle de la protéine IE2 dans l’initiation de l’infection par HHV-6.Human herpesvirus 6 (HHV-6) is an opportunistic pathogen whose infection or reactivation are associated with diseases such as roseola, central nervous system disorders and organ transplant anomalies. Sequencing of the viral genome has exposed the existence of two HHV-6 variants (A and B), with diverging sequences in specific regions, and different biological characteristics. Our work focused on the characterization of HHV-6A immediate-early IE2 protein. Its prompt expression following infection and its transactivating ability suggest that IE2 constitutes a key protein for the establishment of a productive infection, owing to its control over the viral gene expression cascade. Moreover, the IE2 coding transcript is located in the most variable region between HHV-6A and -6B, suggesting that the biology of this protein could help explain the clinical differences between the two viral variants. In order to identify cellular proteins recruited by IE2 during the establishment of infection, we have screened a T-cell library for interaction partners. We have isolated Ubiquitin conjugating enzyme 9 (Ubc9), a protein involved in the small ubiquitin-related modifier (SUMO) conjugation pathway. This interaction has a functional relevance for IE2, with Ubc9 significantly repressing promoter activation by the viral protein. Protein domains essential for IE2 function had never been characterized. We have determined that the N- and C-terminal domains are both required for optimal transactivation, and that the deletion of the C-terminal tail of IE2 significantly alters transactivation and the intranuclear localization of the protein. Moreover, we have determined that the R3 domain of the immediate-early HHV-6A promoter represents an IE2 responsive element. Overall, this work provides a more precise image of the role of IE2 during the initiation of HHV-6 infection and a better comprehension of the biology of this complex virus

Comprehensive characterisation of transcriptional activity during influenza A virus infection reveals biases in cap-snatching of host RNA sequences.

Macrophages in the lung detect and respond to influenza A virus (IAV), determining the nature of the immune response. Using terminal-depth cap analysis of gene expression (CAGE), we quantified transcriptional activity of both host and pathogen over a 24-h time course of IAV infection in primary human monocyte-derived macrophages (MDMs). This method allowed us to observe heterogenous host sequences incorporated into IAV mRNA, "snatched" 5' RNA caps, and corresponding RNA sequences from host RNAs. In order to determine whether capsnatching is random or exhibits a bias, we systematically compared host sequences incorporated into viral mRNA ("snatched") against a complete survey of all background host RNA in the same cells, at the same time. Using a computational strategy designed to eliminate sources of bias due to read length, sequencing depth, and multimapping, we were able to quantify overrepresentation of host RNA features among the sequences that were snatched by IAV. We demonstrate biased snatching of numerous host RNAs, particularly small nuclear RNAs (snRNAs), and avoidance of host transcripts encoding host ribosomal proteins, which are required by IAV for replication. We then used a systems approach to describe the transcriptional landscape of the host response to IAV, observing many new features, including a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation, seen in response to other treatments.IMPORTANCE Infection with influenza A virus (IAV) infection is responsible for an estimated 500,000 deaths and up to 5 million cases of severe respiratory illness each year. In this study, we looked at human primary immune cells (macrophages) infected with IAV. Our method allows us to look at both the host and the virus in parallel. We used these data to explore a process known as "cap-snatching," where IAV snatches a short nucleotide sequence from capped host RNA. This process was believed to be random. We demonstrate biased snatching of numerous host RNAs, including those associated with snRNA transcription, and avoidance of host transcripts encoding host ribosomal proteins, which are required by IAV for replication. We then describe the transcriptional landscape of the host response to IAV, observing new features, including a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation, seen in response to other treatments

A gene expression atlas of the domestic pig

<p>Abstract</p> <p>Background</p> <p>This work describes the first genome-wide analysis of the transcriptional landscape of the pig. A new porcine Affymetrix expression array was designed in order to provide comprehensive coverage of the known pig transcriptome. The new array was used to generate a genome-wide expression atlas of pig tissues derived from 62 tissue/cell types. These data were subjected to network correlation analysis and clustering.</p> <p>Results</p> <p>The analysis presented here provides a detailed functional clustering of the pig transcriptome where transcripts are grouped according to their expression pattern, so one can infer the function of an uncharacterized gene from the company it keeps and the locations in which it is expressed. We describe the overall transcriptional signatures present in the tissue atlas, where possible assigning those signatures to specific cell populations or pathways. In particular, we discuss the expression signatures associated with the gastrointestinal tract, an organ that was sampled at 15 sites along its length and whose biology in the pig is similar to human. We identify sets of genes that define specialized cellular compartments and region-specific digestive functions. Finally, we performed a network analysis of the transcription factors expressed in the gastrointestinal tract and demonstrate how they sub-divide into functional groups that may control cellular gastrointestinal development.</p> <p>Conclusions</p> <p>As an important livestock animal with a physiology that is more similar than mouse to man, we provide a major new resource for understanding gene expression with respect to the known physiology of mammalian tissues and cells. The data and analyses are available on the websites <url>http://biogps.org and http://www.macrophages.com/pig-atlas</url>.</p

Avian Influenza A Virus Polymerase Association with Nucleoprotein, but Not Polymerase Assembly, Is Impaired in Human Cells during the Course of Infection ▿

Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle

Recruitment of RED-SMU1 Complex by Influenza A Virus RNA Polymerase to Control Viral mRNA Splicing

Erratum in PLoS Pathog. 2014 Jul;10(7):e1004317.International audienceInfluenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs

Effect of RED knock-down on the subcellular localization of NP in influenza virus infected cells.

<p>A549 cells were transfected with control non-target (NT) or RED siRNAs, and were subsequently infected with WSN influenza virus at a m.o.i. of 5 pfu/cell. At 5 hpi, cells were fixed, permeabilized, and stained with an antibody specific for the NP protein and with Hoechst 33342. Samples were analyzed under a fluorescence microscope (Inverted Zeiss Observer Z1). <b>A</b>. Representative images of NP localization. <b>B</b>. Percentage of cells with different NP localization. The results of two independent experiments is shown, in which 107 and 94 cells (exp #a), and 164 and 115 cells (exp #b), were scored for the NT and RED siRNA experimental condition, respectively.</p

Detection of ternary PB2-RED-SMU1 complexes.

<p><b>A</b>. The <i>Gaussia princeps</i> luciferase-based complementation assay was performed with the PB2-Gluc1 and Gluc2-SMU1 fusion proteins, in the absence or presence of over-expressed RED protein. The Normalized Luminescence Ratios (NLR) were determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat-1004164-g001" target="_blank">Figure 1A</a>. The dashed line indicates the NLR cut-off that reduces false positive background below 2.5%, as determined using a random reference set of human proteins. The data are expressed as the mean +/− SD of triplicates and are representative of two independent experiments. <b>B</b>. The Gluc2-RED and Gluc1-SMU1 fusion proteins were co-expressed in HEK-293T cells, together with the wild-type PB2 protein or PB2-Strep fusion protein (transfection). Alternatively, HEK-293T cells co-expressing Gluc2-RED and Gluc1-SMU1 were superinfected with the rWSN or rWSN-PB2-Strep viruses (infection). Cell lysates were subjected to purification using StrepTactin beads. Control samples (cells co-expressing Gluc2-RED+Gluc1 or Gluc2+Gluc1-SMU1) were processed in parallel. Luciferase activities and NLRs were determined on an aliquot of the lysates (upper graph) and on the StrepTactin eluates (lower graph) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat-1004164-g001" target="_blank">Figure 1A</a>. The data are expressed as the mean +/− SD of triplicates and are representative of two independent experiments (infection) or a single experiment (transfection).</p

Effect of RED or SMU1 knock-down on the accumulation of influenza virus mRNAs.

<p>A549 cells were transfected with RED (R), SMU1 (S) or control non-target (NT) siRNAs, and were subsequently infected with the WSN virus at a m.o.i. of 5 pfu/cell. Polyadenylated RNAs were purified at 3, 6 and 9 hpi, and RT-qPCR was performed to detect specifically the viral NS1, NS2, M1 and M2 mRNAs, as well as the cellular GAPDH mRNAs. <b>A</b>. Schematic representation of the primers and probes used for RT-qPCR. The NS1, NS2, M1 and M2 mRNAs are depicted. The positions of primers and probes are indicated by black and white arrowheads, respectively, that are oriented according to the sense or antisense orientation of the oligonucleotides. <b>B</b>. The NS1, NS2, M1 and M2 mRNA copy numbers, as determined using the standard curve method and normalized with respect to GAPDH mRNA copy numbers, are shown. The data are expressed as the mean +/− SD of two independent experiments in triplicates, except for the SMU1-siRNA-3 hpi and SMU1-siRNA-6 hpi conditions (one experiment in triplicates). For each mRNA at each time-point, the ratio (in percent) of mRNA copy number in RED- and SMU1-silenced cells to mRNA copy number in NT siRNA-treated cells are indicated below the graphs. Two-way ANOVA was performed to evaluate the effects of RED siRNA treatment. *<b>*</b>: p<0.0001; *: p<0.001; ns: non significant (p>0.05). <b>C</b>. The ratios of NS2/NS1 and M2/M1 mRNA copy numbers in RED- and SMU1-silenced cells are expressed as percentages of the ratios measured in control cells (black bars: 100%). When indicated, two-way ANOVA was performed to evaluate the effect of siRNA treatment. *: p<0.001; ns: non significant (p>0.05).</p

Steady-state levels and subcellular localisation of endogenous RED and SMU1 in infected cells.

<p><b>A</b>. A549 cells were infected at a m.o.i. of 5 pfu/cell with the rWSN virus (+) or mock-infected (−). Total cell extracts were prepared at 0, 3, 6 and 9 hours post-infection (hpi), loaded on a 4–12% SDS-polyacrylamide gel and analyzed by western blotting using antibodies specific for RED, SMU1, NP and GAPDH. <b>B</b>. A549-GFP1-10 cells on coverslips were infected with the rWSN-PB2-GFP11 recombinant virus at a m.o.i. of 5 pfu/cell (WSN), or mock-infected. These conditions allow direct visualization of PB2 in infected cells by trans-complementation of GFP fragments GFP1-10 and GFP11 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat.1004164-Avilov1" target="_blank">[38]</a>. At 6 hpi, cells were fixed, permeabilized, and stained with an antibody specific for the RED or SMU1 protein and with Hoechst 33342. Samples were analyzed under a fluorescence microscope (Inverted Zeiss Observer Z1). Merge views corresponding to RED (red) and SMU1 (cyan), or RED and PB2-GFPcomp (green), are shown. A scale bar is shown.</p