Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (23)
- Nature Immunology
- Nature Biotechnology
- Genes & Development
- Nature Immunology
- PLoS Pathogens
- Current Opinion in Microbiology
- Developmental Cell
- Immunity
- Current Opinion in Microbiology
- Cell
- Cell
- Autophagy
- Proceedings of the National Academy of Sciences of the United States of America
- Current Opinion in Immunology
- PLoS Pathogens
- Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research
- PLoS Pathogens
- PloS One
- Cell Host & Microbe
- Methods in Molecular Biology (Clifton, N.J.)
- Methods in Molecular Biology (Clifton, N.J.)
- Cell Host & Microbe
- Current Biology : CB
Articles by Sara Cherry in JoVE
JoVE Immunology and Infection
RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
Department of Microbiology, Penn Genome Frontiers Institute, University of Pennsylvania
Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.
Other articles by Sara Cherry on PubMed
Entry is a Rate-limiting Step for Viral Infection in a Drosophila Melanogaster Model of Pathogenesis
The identification of host factors that control susceptibility to infection has been hampered by a lack of amenable genetic systems. We established an in vivo model to determine the host factors that control pathogenesis and identified viral entry as a rate-limiting step for infection. We infected Drosophila melanogaster cells and adults with drosophila C virus and found that the clathrin-mediated endocytotic pathway is essential for both infection and pathogenesis. Heterozygosity for mutations in genes involved in endocytosis is sufficient to protect flies from pathogenicity, indicating the exquisite sensitivity and dependency of the virus on this pathway. Thus, this virus model provides a sensitive and efficient approach for identifying components required for pathogenesis.
Temperature-sensitive Control of Protein Activity by Conditionally Splicing Inteins
Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function. Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems. However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use. Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins. We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80. We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.
Genome-wide RNAi Screen Reveals a Specific Sensitivity of IRES-containing RNA Viruses to Host Translation Inhibition
The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.
Host-pathogen Interactions in Drosophila: New Tricks from an Old Friend
Insects rely solely on innate immune responses to combat a wide array of pathogens. With its powerful genetics, drosophila has proven especially powerful for the study of humoral innate immunity, characterized by the rapid induction of antimicrobial peptides. The two signaling pathways involved, Toll and Imd, have been studied intensely, but other aspects of the drosophila immune response are less well understood. A flurry of reports has focused on the mechanisms of phagocytosis, antiviral immunity and viral pathogenesis in drosophila. These studies have taken advantage of genome-wide RNA-mediated interference screening in drosophila cells, as well as more traditional genetic tools available in the fly. This review discusses advances in these exciting new areas of drosophila immunity.
COPI Activity Coupled with Fatty Acid Biosynthesis is Required for Viral Replication
During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.
Genomic RNAi Screening in Drosophila S2 Cells: What Have We Learned About Host-pathogen Interactions?
The détente between pathogen and host has been of keen interest to researchers in spite of being exceedingly difficult to probe. Recently, new RNA interference (RNAi) technologies, in particular in Drosophila tissue culture cells, have made it possible to interrogate the genetics of host organisms rapidly, with nearly complete genomic coverage and high fidelity. Therefore, it is not surprising that the applications of RNAi to the study of host-pathogen interactions were among the first to be published and have already revealed many new insights into the hosts' role in infection. This review will highlight the application of RNAi screening to pathogen-host interactions in Drosophila cells and will reveal some of the lessons learned from this approach.
Dangerous Liaisons: the Apoptotic Engulfment Receptor CED-1 Links Innate Immunity to the Unfolded Protein Response
In this issue of Developmental Cell, Haskins et al. describe a molecular link between an apoptotic cell receptor, a branch of the unfolded protein response, and innate immunity. The finding that the plasma membrane phagocytic receptor CED-1 promotes the expression of unfolded response genes suggests a novel regulatory mechanism for the control of this primitive immune system that may couple the presence of pathogen with the production of secreted antimicrobial effectors.
Autophagy is an Essential Component of Drosophila Immunity Against Vesicular Stomatitis Virus
Intrinsic innate immune mechanisms are the first line of defense against pathogens and exist to control infection autonomously in infected cells. Here, we showed that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, played a direct antiviral role against the mammalian viral pathogen vesicular stomatitis virus (VSV) in the model organism Drosophila. We found that the surface glycoprotein, VSV-G, was likely the pathogen-associated molecular pattern (PAMP) that initiated this cell-autonomous response. Once activated, autophagy decreased viral replication, and repression of autophagy led to increased viral replication and pathogenesis in cells and animals. Lastly, we showed that the antiviral response was controlled by the phosphatidylinositol 3-kinase (PI3K)-Akt-signaling pathway, which normally regulates autophagy in response to nutrient availability. Altogether, these data uncover an intrinsic antiviral program that links viral recognition to the evolutionarily conserved nutrient-signaling and autophagy pathways.
What Have RNAi Screens Taught Us About Viral-host Interactions?
The blossoming of genomic technologies and miniaturization has opened up the field of genomic scale cell-based screening to the study of viral-host interactions. RNAi technology, while still at its infancy, is being used to identify cellular factors required for various viral infections. This has led to the discovery of hundreds of new factors, and has increased our knowledge of the host factors that impact viral infection and highlighted the cellular pathways at play.
Ars2 Links the Nuclear Cap-binding Complex to RNA Interference and Cell Proliferation
Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.
Ars2 Regulates Both MiRNA- and SiRNA- Dependent Silencing and Suppresses RNA Virus Infection in Drosophila
Intrinsic immune responses autonomously inhibit viral replication and spread. One pathway that restricts viral infection in plants and insects is RNA interference (RNAi), which targets and degrades viral RNA to limit infection. To identify additional genes involved in intrinsic antiviral immunity, we screened Drosophila cells for modulators of viral infection using an RNAi library. We identified Ars2 as a key component of Drosophila antiviral immunity. Loss of Ars2 in cells, or in flies, increases susceptibility to RNA viruses. Consistent with its antiviral properties, we found that Ars2 physically interacts with Dcr-2, modulates its activity in vitro, and is required for siRNA-mediated silencing. Furthermore, we show that Ars2 plays an essential role in miRNA-mediated silencing, interacting with the Microprocessor and stabilizing pri-miRNAs. The identification of Ars2 as a player in these small RNA pathways provides new insight into the biogenesis of small RNAs that may be extended to other systems.
VSV Infection is Sensed by Drosophila, Attenuates Nutrient Signaling, and Thereby Activates Antiviral Autophagy
Innate immune mechanisms are the first line of defense against pathogens including viruses. This work identifies autophagy, an innate intracellular degradative pathway, as antiviral against Vesicular Stomatitis Virus (VSV) in Drosophila. VSV is sensed by cells via the surface glycoprotein leading to the attenuation of the nutrient signaling pathway thereby activating an antiviral autophagic program.
The Immune Response Attenuates Growth and Nutrient Storage in Drosophila by Reducing Insulin Signaling
Innate immunity is the primary and most ancient defense against infection. Although critical to survival, coordinating protection against a foreign organism is energetically costly, creating the need to reallocate substrates from nonessential functions, such as growth and nutrient storage. However, the mechanism by which infection or inflammation leads to a reduction in energy utilization by these dispensable processes is not well understood. Here, we demonstrate that activation of the Toll signaling pathway selectively in the fat body, the major immune and lipid storage organ of the fruit fly, Drosophila melanogaster, leads to both induction of immunity and reallocation of resources. Toll signaling in the fat body suppresses insulin signaling both within these cells and non-autonomously throughout the organism, leading to a decrease in both nutrient stores and growth. These data suggest that communication between these two regulatory systems evolved as a means to divert energy in times of need from organismal growth to the acute requirement of combating infection.
Innate Antiviral Immunity in Drosophila
The study of Drosophila, and other genetically tractable insects, has expanded our understanding of innate immunity and more recently antiviral innate mechanisms. The Drosophila antiviral program includes inflammatory signaling cascades as well as antiviral RNA silencing and autophagy. This review will highlight the recent discoveries in antiviral immunity in insects and will reveal some of the lessons learned.
A Kinome RNAi Screen Identified AMPK As Promoting Poxvirus Entry Through the Control of Actin Dynamics
Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator.
Leucine-rich Repeat (in Flightless I) Interacting Protein-1 Regulates a Rapid Type I Interferon Response
The cell autonomous response to viral infection is carefully regulated to induce type I interferons (IFNs), which in turn induce the establishment of an antiviral state. Leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) and LRRFIP2 are 2 related proteins that have been identified as interacting with MyD88 and Flightless I homolog, a leucine-rich repeat protein. LRRFIP2 positively regulates NFκB and macrophage cytokine production after lipopolysaccharide, but less is known about LRRFIP1. We hypothesized that LRRFIP1 could be more important in antiviral responses, as overexpression led to type I IFN production in a pilot study. The induction of type I IFNs occurred even in the absence of virus, but was enhanced by the presence of virus. Conversely, knockdown of LRRFIP1 compromised IFN expression. We found that LRRFIP1 was rapidly recruited to influenza-containing early endosomes in a p38-dependent fashion. This was specific for virus-containing endosomes as there was almost no colocalization of LRRFIP1 with early endosomes in the absence of virus. Further, LRRFIP1 was recruited to RNA-containing vesicles. Taken together, these data suggest that LRRFIP1 participates in cell responses to virus at early time points and is important for type I IFN induction.
Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells
Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.
Rift Valley Fever Virus Infection of Human Cells and Insect Hosts is Promoted by Protein Kinase C Epsilon
As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.
Comparative RNAi Screening Reveals Host Factors Involved in Enterovirus Infection of Polarized Endothelial Monolayers
Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through the blood brain barrier (BBB) endothelium to cause aseptic meningitis. To identify cellular components required for CVB and PV infection of human brain microvascular endothelial cells, an in vitro BBB model, we performed comparative RNAi screens and identified 117 genes that influenced infection. Whereas a large proportion of genes whose depletion enhanced infection (17 of 22) were broadly antienteroviral, only 46 of the 95 genes whose depletion inhibited infection were required by both CVB and PV and included components of cell signaling pathways such as adenylate cyclases. Downregulation of genes including Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases displayed specificity in their requirement for either CVB or PV infection. These findings highlight the pathways hijacked by enteroviruses for entry and replication in the BBB endothelium, a specialized and clinically relevant cell type for these viruses.
RNAi Screening for Host Factors Involved in Viral Infection Using Drosophila Cells
Since viral pathogens represent a significant threat to human health, a better understanding of the cellular factors that impact infection would facilitate the development of therapeutics. The recent advent of RNA interference (RNAi) technology coupled with the ease and efficiency of RNAi in Drosophila cell culture has led to the widespread use of this experimental system for high-throughput RNAi screening of host factors required for viral infection [Cherry et al., Genes Dev 19:445-452, 2005; Hao et al., Nature 45:890-893, 2008; Sessions et al., Nature 458:1047-1050, 2009]. Here, we describe the use of this system for the identification of host factors that impact viral infection.
RNAi Screening in Mammalian Cells to Identify Novel Host Cell Molecules Involved in the Regulation of Viral Infections
It is clear that viral entry, replication, and spread is a complex process involving a dialog between the virus and the targeted host cell. Viruses have evolved highly specific strategies to hijack cellular factors to promote their internalization, initiate their replication, and facilitate their eventual spread. However, the identification of many of these host cell molecules has been hindered by the requirement for robust genome-scale loss-of-function assays that are capable of targeting a wide variety of host factors. The more recent use of genome-scale or genome-wide RNA interference (RNAi) screens have extended our knowledge of the complex interplay between a virus and host and have implicated a wide variety of cellular factors required for infection of a number of viruses. Here, we describe an approach to target mammalian host cell factors involved in regulating viral infections by the use of a genome-scale RNAi library screen.
Natural Resistance-associated Macrophage Protein is a Cellular Receptor for Sindbis Virus in Both Insect and Mammalian Hosts
Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection.
The Exoribonuclease Nibbler Controls 3' End Processing of MicroRNAs in Drosophila
MicroRNAs (miRNAs) are endogenous noncoding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation [1-3]. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription [4-8], processing [9-11], and stability [12, 13], with miRNA deregulation linked with diseases [14] and neurodegenerative disorders [15]. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage [16-18] to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute1 (Ago1) within the RNA-induced silencing complex (RISC) [19, 20]. Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3' end, suggesting a novel biogenesis mechanism involving 3' end processing. To define the cellular factors responsible, we performed an RNA interference (RNAi) screen and identified a putative 3'→5' exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34. Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings suggest that Nbr-mediated 3' end processing represents a critical step in miRNA maturation that impacts miRNA diversity.
