Ribonucleoprotein transport in Negative Strand RNA viruses.

Negative-sense, single-stranded RNA (-ssRNA) viruses comprise some of the deadliest human pathogens (Ebola, rabies, influenza A viruses etc.). Developing therapeutic tools relies on a better understanding of their multiplication cycle. For these viruses, the genome replication and transcription activities most-often segregate in membrane-less environments called inclusion bodies (IBs) or viral factories. These "organelles" usually locate far from the cell surface from where new virions are released, and -ssRNA viruses do not encode for transport factors. The efficient trafficking of the genome progeny toward the cell surface is most often ensured by mechanisms co-opting the cellular machineries. In this review, for each -ssRNA viral family, we cover the methods employed to characterize these host-virus interactions, the strategies used by the viruses to promote the virus genome transport, and the current gaps in the literature. Finally, we highlight how Rab11 has emerged as a target of choice for the intracellular transport of -ssRNA virus genomes.

Monomethyl branched-chain fatty acids are critical for Caenorhabitis elegans survival in elevated glucose conditions.

The maintenance of optimal membrane composition under basal and stress conditions is critical for the survival of an organism. High-glucose stress has been shown to perturb membrane properties by decreasing membrane fluidity, and the membrane sensor PAQR-2 is required to restore membrane integrity. However, the mechanisms required to respond to elevated dietary glucose are not fully established. In this study, we used a 13C stable isotope-enriched diet and mass spectrometry to better understand the impact of glucose on fatty acid dynamics in the membrane of Caenorhabditis elegans. We found a novel role for monomethyl branched-chain fatty acids (mmBCFAs) in mediating the ability of the nematodes to survive conditions of elevated dietary glucose. This requirement of mmBCFAs is unique to glucose stress and was not observed when the nematode was fed elevated dietary saturated fatty acid. In addition, when worms deficient in elo-5, the major biosynthesis enzyme of mmBCFAs, were fed Bacillus subtilis (a bacteria strain rich in mmBCFAs) in combination with high glucose, their survival rates were rescued to wild-type levels. Finally, the results suggest that mmBCFAs are part of the PAQR-2 signaling response during glucose stress. Taken together, we have identified a novel role for mmBCFAs in stress response in nematodes and have established these fatty acids as critical for adapting to elevated glucose.

Hardening of Respiratory Syncytial Virus Inclusion Bodies by Cyclopamine Proceeds through Perturbation of the Interactions of the M2-1 Protein with RNA and the P Protein.

Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. The assembly of IBs is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We showed that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1-P interaction, thereby affecting the relative mobility of the proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.

CeFra-seq reveals broad asymmetric mRNA and noncoding RNA distribution profiles in Drosophila and human cells.

Cells are highly asymmetrical, a feature that relies on the sorting of molecular constituents, including proteins, lipids, and nucleic acids, to distinct subcellular locales. The localization of RNA molecules is an important layer of gene regulation required to modulate localized cellular activities, although its global prevalence remains unclear. We combine biochemical cell fractionation with RNA-sequencing (CeFra-seq) analysis to assess the prevalence and conservation of RNA asymmetric distribution on a transcriptome-wide scale in Drosophila and human cells. This approach reveals that the majority (∼80%) of cellular RNA species are asymmetrically distributed, whether considering coding or noncoding transcript populations, in patterns that are broadly conserved evolutionarily. Notably, a large number of Drosophila and human long noncoding RNAs and circular RNAs display enriched levels within specific cytoplasmic compartments, suggesting that these RNAs fulfill extra-nuclear functions. Moreover, fraction-specific mRNA populations exhibit distinctive sequence characteristics. Comparative analysis of mRNA fractionation profiles with that of their encoded proteins reveals a general lack of correlation in subcellular distribution, marked by strong cases of asymmetry. However, coincident distribution profiles are observed for mRNA/protein pairs related to a variety of functional protein modules, suggesting complex regulatory inputs of RNA localization to cellular organization.

Optimized FISH methods for visualizing RNA localization properties in Drosophila and human tissues and cultured cells.

Eukaryotic gene expression is orchestrated by a large number of regulatory steps to modulate the synthesis, maturation and fate of various families of protein-coding and non-coding RNA molecules. Defining the subcellular localization properties of an RNA molecule is thus of considerable importance for gleaning its function(s) and for elucidating post-transcriptional gene regulation pathways. For decades, fluorescent In Situ hybridization (FISH) has constituted the gold-standard technique for assessing RNA expression and distribution properties in cultured cells, tissue specimens, and whole mount organisms. Recently, several attempts aimed at advancing multiplex RNA-FISH experiments have been published. However, these procedures are both financially demanding and technically challenging, while their full potential remains unexploited. Here we describe an optimized RNA-FISH method employing the Tyramide Signal Amplification system that robustly enhances resolution and sensitivity needed for exploring RNA localization in Drosophila embryos, tissues and commonly cultured human and insect cell lines. Methodological details and key parameters are outlined for high-throughput analyses conducted in 96-well plate format.

Exploration of binary virus-host interactions using an infectious protein complementation assay.

A precise mapping of pathogen-host interactions is essential for comprehensive understanding of the processes of infection and pathogenesis. The most frequently used techniques for interactomics are the yeast two-hybrid binary methodologies, which do not recapitulate the pathogen life cycle, and the tandem affinity purification mass spectrometry co-complex methodologies, which cannot distinguish direct from indirect interactions. New technologies are thus needed to improve the mapping of pathogen-host interactions. In the current study, we detected binary interactions between influenza A virus polymerase and host proteins during the course of an actual viral infection, using a new strategy based on trans-complementation of the Gluc1 and Gluc2 fragments of Gaussia princeps luciferase. Infectious recombinant influenza viruses that encode a Gluc1-tagged polymerase subunit were engineered to infect cultured cells transiently expressing a selected set of Gluc2-tagged cellular proteins involved in nucleocytoplasmic trafficking pathways. A random set and a literature-curated set of Gluc2-tagged cellular proteins were tested in parallel. Our assay allowed the sensitive and accurate recovery of previously described interactions, and it revealed 30% of positive, novel viral-host protein-protein interactions within the exploratory set. In addition to cellular proteins involved in the nuclear import pathway, components of the nuclear pore complex such as NUP62 and mRNA export factors such as NXF1, RMB15B, and DDX19B were identified for the first time as interactors of the viral polymerase. Gene silencing experiments further showed that NUP62 is required for efficient viral replication. Our findings give new insights regarding the subversion of host nucleocytoplasmic trafficking pathways by influenza A viruses. They also demonstrate the potential of our infectious protein complementation assay for high-throughput exploration of influenza virus interactomics in infected cells. With more infectious reverse genetics systems becoming available, this strategy should be widely applicable to numerous pathogens.

Bacterial diet modulates tamoxifen-induced death via host fatty acid metabolism.

Tamoxifen is a selective estrogen receptor (ER) modulator that is used to treat ER-positive breast cancer, but that at high doses kills both ER-positive and ER-negative breast cancer cells. We recapitulate this off-target effect in Caenorhabditis elegans, which does not have an ER ortholog. We find that different bacteria dramatically modulate tamoxifen toxicity in C. elegans, with a three-order of magnitude difference between animals fed Escherichia coli, Comamonas aquatica, and Bacillus subtilis. Remarkably, host fatty acid (FA) biosynthesis mitigates tamoxifen toxicity, and different bacteria provide the animal with different FAs, resulting in distinct FA profiles. Surprisingly these bacteria modulate tamoxifen toxicity by different death mechanisms, some of which are modulated by FA supplementation and others by antioxidants. Together, this work reveals a complex interplay between microbiota, FA metabolism and tamoxifen toxicity that may provide a blueprint for similar studies in more complex mammals.

WormPaths: Caenorhabditis elegans metabolic pathway annotation and visualization.

In our group, we aim to understand metabolism in the nematode Caenorhabditis elegans and its relationships with gene expression, physiology, and the response to therapeutic drugs. Visualization of the metabolic pathways that comprise the metabolic network is extremely useful for interpreting a wide variety of experiments. Detailed annotated metabolic pathway maps for C. elegans are mostly limited to pan-organismal maps, many with incomplete or inaccurate pathway and enzyme annotations. Here, we present WormPaths, which is composed of two parts: (1) the careful manual annotation of metabolic genes into pathways, categories, and levels, and (2) 62 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on the WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the future, we envision further developing these maps to be more interactive, analogous to road maps that are available on mobile devices.

WormPaths: Caenorhabditis elegans metabolic pathway annotation and visualization.

In our group, we aim to understand metabolism in the nematode Caenorhabditis elegans and its relationships with gene expression, physiology and the response to therapeutic drugs. On March 15, 2020, a stay-at-home order was put into effect in the state of Massachusetts, USA, to flatten the curve of the spread of the novel SARS-CoV2 virus that causes COVID-19. For biomedical researchers in our state, this meant putting a hold on experiments for nine weeks until May 18, 2020. To keep the lab engaged and productive, and to enhance communication and collaboration, we embarked on an in-lab project that we all found important but that we never had the time for: the detailed annotation and drawing of C. elegans metabolic pathways. As a result, we present WormPaths, which is composed of two parts: 1) the careful manual annotation of metabolic genes into pathways, categories and levels, and 2) 66 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on our WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the unfortunate event of additional lockdowns, we envision further developing these maps to be more interactive, with an analogy of road maps that are available on mobile devices.

Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs.

Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19.