Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation

Genomic location of the major ribosomal protein gene locus determines Vibrio cholerae global growth and infectivity.

International audienceThe effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, localize near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fast growth or facilitate the assembly of ribosomes and transcription foci by keeping physically close the many components of these macromolecular machines. To explore this, we used novel recombineering tools to create a set of Vibrio cholerae strains in which S10-spec-α (S10), a locus bearing half of the ribosomal protein genes, was systematically relocated to alternative genomic positions. We show that the relative distance of S10 to the origin of replication tightly correlated with a reduction of S10 dosage, mRNA abundance and growth rate within these otherwise isogenic strains. Furthermore, this was accompanied by a significant reduction in the host-invasion capacity in Drosophila melanogaster. Both phenotypes were rescued in strains bearing two S10 copies highly distal to oriC, demonstrating that replication-dependent gene dosage reduction is the main mechanism behind these alterations. Hence, S10 positioning connects genome structure to cell physiology in Vibrio cholerae. Our results show experimentally for the first time that genomic positioning of genes involved in the flux of genetic information conditions global growth control and hence bacterial physiology and potentially its evolution

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila

International audienceHow persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA virus in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence. The most well-characterized viral infections are those with human or economic effects. However, regardless of the organism under consideration, there are viruses able to infect that organism. Viral fossil registers highlight the long coevolutionary history between virus an

Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects

International audienceTransgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline

S10-positioning influences <i>D</i>. <i>melanogaster</i> infection-capacity of <i>V</i>. <i>cholerae</i>.

<p>Bacterial load within flies 48h pi with parental (n = 53), S10Tnp-35 (n = 21), S10Tnp-510 (n = 24), S10Tnp-1120 (n = 40), S10TnpC2+479(n = 21), ΔS10Tnp-510 (n = 21), ΔS10Tnp-1120 (n = 15), ΔS10TnpC2+479 (n = 11), S10Md(-510;-1120) (n = 15) or S10Md(-1120;C2+479) (n = 15) strains are shown. When the observed value was 0 CFU/fly points were plotted as 10⁰. Statistical significance was analyzed using Kruskal-Wallis non-parametric tests followed by Dunn’s multiple comparisons using parental as control respectively. n.s. stands for non-significant, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001.</p

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila

International audienceHow persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA virus in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence. The most well-characterized viral infections are those with human or economic effects. However, regardless of the organism under consideration, there are viruses able to infect that organism. Viral fossil registers highlight the long coevolutionary history between virus an

S10 dosage and expression diminish in a distance-related manner correlating with GR reduction.

<p><b>(a)</b> Expected trend on S10/ter1 and ori1/S10 ratios according to locus repositioning. Ellipses represent chromosomes. Colored dots depict <i>oriC1</i> and <i>oriC2</i> and termini of Chr1 (<i>ter1</i>) and Chr2 (<i>ter2</i>). Simultaneous replication rounds are shown. An orange arrow represents the S10 locus. The expected trend for ori1/s10 and s10/ter1 is shown by top and bottom triangles. <b>(b)</b> Gene dosage measurements obtained by qPCR in fast-growth conditions. <b>(c)</b> S10 expression normalized to parental strains obtained by RT-qPCR. <b>b</b> and <b>c</b> show the mean and error bars representing 95% CI. Statistical significance was assessed by one-way ANOVA two tailed test and Tukey test for multiple comparisons. n.s. stands for non-significant, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001. <b>(d)</b> S10 dosage (red), expression (green) and % variation of μ (blue) of each bacterial strain were plotted as a function S10 position within the genome measured as % of replichore length. Linear regression is plotted for each variable. Chr2 was overlapped to Chr1 according to cell cycle order. Chromosomes are schematized on the top of the graph.</p

GR defect is consequence of gene dosage reduction.

<p>S10 dosage effect was quantified by averaging obtained μ for each strain and normalizing it to the value of the parental strain. Results are expressed as percentage of the variation (μ %) with 95% CI showing complementation of S10Tnp-1120 mutant. Values were obtained from 5 experiments using several independently obtained clones. Statistical significance was assessed by one-way ANOVA two-tailed test. Tukey test was performed for multiple comparisons. n.s. stands for non-significant, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001.</p

Gene dosage measurements performed by qPCR experiments on the full strain set.

<p>Gene dosage measurements performed by qPCR experiments on the full strain set.</p

Generation of S10Tnp strains.

<p><b>(a)</b> S10 is moved by flanking it by HK022 attL and attR sites. Upon transient expression of phage recombinases a DNA circle containing S10 is excised and <i>bla</i> reporter is reconstituted. Viable carbenicillin resistant (Carb^R) cells are obtained if S10 reintegrates at attB’. <b>(b)</b> The obtained S10Tnp strains: ellipses represent chromosomes while small dots represent origin of replication of Chr1 (<i>oriC1</i>,red) and Chr2 (<i>oriC2</i>, blue). Orange arrows depict S10 position within the genome. The green arrow shows <i>bla</i>. Left panel, picture representative of one of the parental strains from which S10Tnp derivatives were produced using attB’ sites at different positions. Right panel, the derivatives showing S10 relocation are shown. Insets correspond to CLSM images of each strain stained with FM5-95, the white bar represents 5 μm (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005156#sec014" target="_blank">Supporting Information</a>).</p