Drosophila rnai screen identifies host genes important for influenza virus

Author information Copyright and License information Disclaimer. Correspondence to: Yoshihiro Kawaoka 2, 4, 5, 6 and Paul Ahlquist 1 Correspondence and requests for materials should be addressed to Y.

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Go to:. Open in a separate window. Renilla luciferase and cell viability assays Renilla luciferase activity and cell viability were measured with established Renilla luciferase and CellTiter-Glo assay systems Promega according to the manufacturer's instructions; signals were read with a GLOMAX 96 microplate luminometer.

Inhibitor treatment of mammalian cells Mitochondrial electron transport chain inhibitors were used at the following final concentrations: rotenone 0. Click here to view. Kuttenkeuler D, Boutros M. Genome-wide RNAi as a route to gene function in Drosophila.

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Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes. J Virol.

Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency. Perez L, Carrasco L. J Gen Virol. Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses. Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice. Momose F, et al. Satterly N, et al. Influenza virus targets the mRNA export machinery and the nuclear pore complex. Stevens TH, Forgac M. Annu Rev Cell Dev Biol. Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase.

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Wurzer WJ, et al. Caspase 3 activation is essential for efficient influenza virus propagation. Embo J. Apoptosis: a mechanism of cell killing by influenza A and B viruses.

The possible role of cytochrome c oxidase in stress-induced apoptosis and degenerative diseases. Biochim Biophys Acta. He TC, et al. A simplified system for generating recombinant adenoviruses.

Isolation of cell lines that show novel, murine leukemia virus-specific blocks to early steps of retroviral replication. Ge Q, et al. Dave RS, et al. Full text links Read article at publisher's site DOI : Smart citations by scite. The number of the statements may be higher than the number of citations provided by EuropePMC if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.

Explore citation contexts and check if this article has been supported or disputed. Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy. Discovering antiviral restriction factors and pathways using genetic screens. Data Data behind the article This data has been text mined from the article, or deposited into data resources. BioStudies: supplemental material and supporting data.

Data that cites the article This data has been provided by curated databases and other sources that have cited the article. Faculty Opinions. Similar Articles To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. A host susceptibility gene, DR1, facilitates influenza A virus replication by suppressing host innate immunity and enhancing viral RNA replication.

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Genome-wide RNAi screen for viral replication in mammalian cell culture. A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens.

Cellular networks involved in the influenza virus life cycle. Using RNA interference to target cellular factors involved in protein trafficking, it was revealed that Nef-dependent CD4 downregulation required a specific interaction with AP2, a complex involved in clathrin-mediated endocytosis, but not other AP complexes.

This discovery was followed up using HeLa cells where it was shown that the Nef-AP2 interaction is functionally conserved in humans. Another study used transgenic Drosophila to show that Nef expression in larval wing discs also caused apoptosis through activation of the conserved JNK signaling pathway Lee et al. These findings may help to explain how Nef expression during HIV infection contributes to the decline of T-cell immune function that is characteristic of AIDS progression.

Tat is an HIV-1 protein required for viral gene expression and is essential for viral replication. Tat was expressed in transgenic Drosophila , where it disrupted microtubule polymerization and kinetochore dynamics via a direct interaction with tubulin Battaglia et al.

Ensuing research in human cells validated the importance of this finding that helped to advance the understanding of the mechanisms of HIV pathogenesis Butler et al. Tat was previously shown to localize to nucleoli in human cells, but the function of Tat in the nucleolus was unclear. Another study demonstrated that expression of Tat protein in the Drosophila ovary showed nucleolar localization Ponti et al.

In these transgenic females, Tat was shown to affect the maturation of ribosomes through the inhibition of rRNA processing, which resulted in a reduced number of ribosomes in the cytoplasm. Many viruses regulate protein production to facilitate viral replication and to modulate the apoptotic response of the host cell, so this research suggests a mechanism by which Tat may play a role in HIV-1 pathogenesis. Rev was studied in cultures of Drosophila S2 cells through the use of a Rev gene co-transfected with a plasmid containing a copy of the viral env gene Ivey-Hoyle and Rosenberg, It was found that Rev acts in Drosophila cells as it does in mammalian cells by promoting the transport of env mRNA from the nucleus to the cytoplasm.

This suggests that the Rev protein functions by interacting with host cellular factors that are conserved between humans and insects Brighty and Rosenberg, Future research will benefit from using Drosophila to study HIV protein function due to the high conservation between insect and human cellular pathways.

However, we believe that this tool remains underutilized and holds great potential for the study of other human viruses. Viruses that would make good candidates for future study in Drosophila would include those that have a large impact on human populations.

A small viral genome would allow for a simpler selection of candidate genes for further study. For example, a comparison of the functional differences between genes of the different strains could help to uncover what makes one strain more pathogenic than another.

Based on these criteria, we have identified three candidates — the human papillomavirus, the hepatitis C virus, and the yellow fever virus — which could potentially benefit from studies using Drosophila as a model. We anticipate that in the future D. We are also grateful to Dr. Brian Ceresa for helpful comments during the preparation of the manuscript.

National Center for Biotechnology Information , U. Published online Dec Tamara T. Hughes , Amanda L. Allen , Joseph E. Bardin , Megan N. Christian , Kansei Daimon , Kelsey D. Dozier , Caom L. Hansen , Lisa M. Author information Article notes Copyright and License information Disclaimer. All rights reserved. Elsevier hereby grants permission to make all its COVIDrelated research that is available on the COVID resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source.

This article has been cited by other articles in PMC. Abstract Viruses are infectious particles whose viability is dependent on the cells of living organisms, such as bacteria, plants, and animals. Introduction Viral infection is associated with a number of diseases ranging from the common cold to cancer.

Table 1 Human viruses studied using Drosophila melanogaster. Open in a separate window. Studies of human viruses using D. HIV More than 30 million individuals are infected with the human immunodeficiency virus HIV worldwide, resulting in about 2 million deaths annually Kilmarx, Future directions D. References Adamson A. EMBO J. A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response.

PLoS Pathog. Cell Sci. PLoS Negl. A cis-acting repressive sequence that overlaps the Rev-responsive element of human immunodeficiency virus type 1 regulates nuclear retention of env mRNAs independently of known splice signals.

Neurodegenerative effects of recombinant HIV-1 Tat 1—86 are associated with inhibition of microtubule formation and oxidative stress-related reductions in microtubule-associated protein-2 a, b Neurochem. Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. Rev-GFP transgenic lines for studies of nucleocytoplasmic transport in Drosophila.

The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function. Cell Biol. Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor. HIV-1 Tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic Bcl-2 relative Bim.

Role of two-way airflow owing to temperature difference in severe acute respiratory syndrome transmission: revisiting the largest nosocomial severe acute respiratory syndrome outbreak in Hong Kong.

Structural insights into the retroviral DNA integration apparatus. VSV infection is sensed by Drosophila , attenuates nutrient signaling, and thereby activates antiviral autophagy. Transgenic inhibitors of RNA interference in Drosophila. Fly Austin ; 1 — The E2F transcriptional network: old acquaintances with new faces. GAL4 system in Drosophila : a fly geneticist's Swiss army knife.

A Drosophila model of Parkinson's disease. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. The native Wolbachia endosymbionts of Drosophila melanogaster and Culex quinquefasciatus increase host resistance to West Nile virus infection. PLoS One. Drosophila RNAi screen identifies host genes important for influenza virus replication. Human gamma protein results in abnormal cell proliferation in the developing eye of Drosophila melanogaster.

Cell Div. Rev-dependent expression of human immunodeficiency virus type 1 gp in Drosophila melanogaster cells. In total, knockdown of 11 genes significantly reduced NP expression and interfered with nuclear export of NP.

Samples were stained for nuclei blue and NP green. Data show mean plus s. Samples were stained for influenza virus green and CD63 red. Images are representative of three independent experiments in a and c. Most of the analysed targets had no effect on virus cell entry, as indicated by robust vRNA detection Fig.

This demonstrates that virus propagation is affected at a stage between virus entry and mRNA synthesis. Accordingly, considerably fewer virus particles co-localized with CDlabelled late endosomes upon SON knockdown Fig. Consistent with its reported antiviral 8 and proviral functions 13 , these seemingly contradictory results indicate that NUP98 exerts an inhibitory effect early in the life cycle but is mandatory for completion of viral replication.

Taken together, these data reveal that the 11 targets identified as reducing NP expression levels interfere with early events in virus replication. In contrast, the remaining seven factors analysed in this set of experiments, such as CLK1 or p27 CDKN1B , probably exert their function during later infection stages.

To mimic in vivo conditions more closely, we tested the effect of target knockdown on influenza virus replication in primary normal human bronchial epithelial cells NHBE. Immunoblot analysis corroborated our qRT—PCR results, revealing markedly reduced M2 protein levels following treatment with TG, whereas M1 protein levels remained relatively constant Fig.

Interestingly, replication of vesicular stomatitis virus VSV , which, unlike influenza, does not depend on splicing of its own viral RNA, was only slightly reduced in the presence of TG Supplementary Fig. Data in a — c are mean plus standard deviation s. Blots in d are representative of three independent experiments. During the primary screen and the hit validation, knockdown of the cell cycle regulator p27 led to a strong inhibition of influenza virus replication.

The observation that a lack of p27 reduces influenza virus replication in vivo but does not affect mouse viability indicates that certain cellular proteins involved in influenza virus replication are dispensable for the host organism.

This genome-wide RNAi screen in human cells for factors affecting influenza virus replication has provided new and comprehensive information on host cell determinants of replication, and uncovered potential targets for novel antiviral strategies.

We provide in vitro and in vivo evidence for the role of CLK1 and the tumour suppressor p27, using a small molecule inhibitor and a homozygous knockout model, respectively. Most of the hits analysed in-depth seem to function during early infection processes such as viral protein synthesis and nuclear export of viral RNA. Importantly, most of the validated hits are essential for a broad spectrum of influenza viruses, including the pandemic swine-origin H1N1 influenza virus and even a highly pathogenic avian H5N1 strain.

This holds promise for the therapeutic potential of these targets against novel emerging influenza viruses with minimized likelihood of developing drug-resistant variants. Transient interference with distinct host cell functions during infection is likely to extend our current armament, consisting of vaccines and virus-targeted drugs, in the battle against the recurring threat of seasonal and pandemic influenza virus infections.

Supplements added at 0. Cells were regularly checked for mycoplasma contamination by PCR. Virus stocks were titrated by standard plaque assay on MDCK cells using an agar overlay medium The number of automatically counted nuclei was further used to estimate cytotoxic effects of specific siRNAs.

The siRNA was classified as being toxic if or fewer nuclei were determined within one well of a well plate. To quantify infectious viruses in the supernatants of siRNA-transfected A cells during the primary RNAi screen, we used a luciferase-based reporter system. Luciferase expression is therefore only detectable in the presence of the viral polymerase, thus allowing quantification of infectious viruses.

All siRNAs were purchased from Qiagen. For western blot experiments, siRNA transfection was carried out in well plates. Cells were fixed with 3.

The numbers of influenza-infected and host cells were determined using an automated microscope Olympus Soft Imaging Solutions. For determination of NP localization, mean and total intensities of NP were analysed. NP located within the same area as the Hoechst staining was defined as nuclear NP. NP located within a 5-pixel-wide ring around the nuclei was defined as cytosolic NP.

For each experiment identical camera settings were used. To quantify infectious virus particles in infected cell culture supernatants, 5, or 12, MDCK cells were seeded in or well plates, respectively. This script applies the R-package GOstats developed by ref.

Briefly, we defined a gene universe consisting of 22, genes contained and annotated in the genome-wide library and processed the hit list against this universe with respect to molecular function MF , cellular component CC and biological process BP.

Each Gene Ontology term is associated with X number of genes, providing a relative frequency A. In the hit list, the same term is connected to Y genes giving a relative frequency B. B divided by A is the enrichment factor. Fusion between influenza viruses and cellular endosomes was detected using confocal microscopy. Staining was performed with ECL western blotting detection reagent Amersham. Band intensities were determined using the Aida image analyser V. All experiments were done in triplicate.

Briefly, one day before transfection, 3, cells per well were seeded onto a well plate. Knockdown measurements were performed independently three times. The relative expression levels of target mRNA were normalized against control transfected cells.

GAPDH was used as an internal standard. The amount of infectious viruses in the supernatant was quantified using the replication assay see above. For identification of primary hits, three parameters were included: luciferase expression, the percentage of infected cells as determined by immunofluorescence microscopy, and the total number of infected cells. The latter parameter was informative because the number of viruses per well correlated with the number of infected cells, with minor influence of cells present.

To maximize the robustness of the hit selection and to minimize false positives owing to off-target effects, raw screening data from all three parameters were subjected separately to an analysis pipeline incorporating statistical checkpoints at each step Supplementary Fig. First, we excluded non-expressed genes by determining constitutive or inducible expression via microarray profiling of non-infected and infected A samples 5, genes were not expressed. Third, non-toxic siRNAs targeting expressed genes were further analysed.

For statistical analysis of luciferase assay data obtained from the genome-wide screen, the following plate-wise quality control criteria were used: 1 the average signal from the non-targeting control wells Allstars was greater than 10, counts; 2 the difference in signal strength between the non-targeting control Allstars ; and 3 the inhibitory control NP was at least two orders of magnitude.

The revised raw data were subjected to statistical analysis using cellHTS 31 , an R-implemented software package for the analysis of cell-based high-throughput RNAi screen data. Raw data were normalized using the B-score method to further exclude positional effects Next, a Z -score transformation was applied to centre and scale the plate-wise data.

The medians of the centred and scaled values of at least three independent replicates were used for redundant siRNA activity RSA analysis 33 , which applies a rank-based hypergeometric distribution test to identify hits.

Only genes for which two corresponding siRNAs were scored as hits were analysed further. For analysis of hit validation data, the normalized per cent inhibition of infectious virus particles was calculated for each siRNA. Briefly, the difference of each sample value subtracted from the median of the non-targeting control Allstars values of the particular plate was divided by the difference of the medians of the non-targeting control and the inhibitory control siNP.

Genes were scored as validated hits if at least two siRNAs, which did not impair cell viability, fulfilled this criterion. The samples sizes are individually defined as the number of main objects per well detected by the automated image analysis package ScanR. Horimoto, T. Influenza: lessons from past pandemics, warnings from current incidents.

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Trends Mol. Lutz, A. Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication.