Protein Complementation Assay Research Articles

Abstract 2057: Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay

Abstract The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon its generation via cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, it binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given its significant role as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. Credible evidence suggests that the cGAS–cGAMP–STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of Protein Complementation Assay (PCA) using nanoluciferase (nanoluc) complementation technology where a small peptide (SmBiT) derived from nanoluc linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. We have optimized the assay for higher sensitivity down to less than 10 nM cGAMP and less than one ng of cGAS and thus, and it is suitable for testing compounds that modulate cGAS activity. We also improved cell lysis condition to increase recovery of cGAMP that is generated intracellularly using several cGAS activators and correlating its concentration with cytokine release. We envision using this assay to monitor cGAMP presence in sera and spinal fluids of patients who have ALS and Alzheimer diseases. The assay is homogenous, with minimal or no false hits using LOPAC screening and HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, kevin Hsiao, Nate Murray, dareen Mikheil, Matt Larsen, Hui Wang. Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2057.

Novel Antiviral Molecules against Ebola Virus Infection.

Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.

Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay.

Background: Adenosine A1 receptor (A1AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep-wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A1AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A1AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A1AR-mediated β-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit® technology, a protein complementation assay was developed in which the A1AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of β-arrestin 2. Stimulation of A1AR results in the recruitment of β-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay. Results: The assay gives highly reproducible results with a very good signal-to-noise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of β-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A1AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A1AR-mediated recruitment of β-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A1AR-mediated β-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract.

Detecting and measuring of GPCR signaling - comparison of human induced pluripotent stem cells and immortal cell lines.

G protein-coupled receptors (GPCRs) are a large family of transmembrane proteins that play a major role in many physiological processes, and thus GPCR-targeted drug development has been widely promoted. Although research findings generated in immortal cell lines have contributed to the advancement of the GPCR field, the homogenous genetic backgrounds, and the overexpression of GPCRs in these cell lines make it difficult to correlate the results with clinical patients. Human induced pluripotent stem cells (hiPSCs) have the potential to overcome these limitations, because they contain patient specific genetic information and can differentiate into numerous cell types. To detect GPCRs in hiPSCs, highly selective labeling and sensitive imaging techniques are required. This review summarizes existing resonance energy transfer and protein complementation assay technologies, as well as existing and new labeling methods. The difficulties of extending existing detection methods to hiPSCs are discussed, as well as the potential of hiPSCs to expand GPCR research towards personalized medicine.

Evidence and Metabolic Implications for a New Non-Canonical Role of Cu-Zn Superoxide Dismutase

Copper-zinc superoxide dismutase 1 (SOD1) has long been recognized as a major redox enzyme in scavenging superoxide radicals. However, there is little information on its non-canonical role and metabolic implications. Using a protein complementation assay (PCA) and pull-down assay, we revealed novel protein-protein interactions (PPIs) between SOD1 and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) or epsilon (YWHAE) in this research. Through site-directed mutagenesis of SOD1, we studied the binding conditions of the two PPIs. Forming the SOD1 and YWHAE or YWHAZ protein complex enhanced enzyme activity of purified SOD1 in vitro by 40% (p < 0.05) and protein stability of over-expressed intracellular YWHAE (18%, p < 0.01) and YWHAZ (14%, p < 0.05). Functionally, these PPIs were associated with lipolysis, cell growth, and cell survival in HEK293T or HepG2 cells. In conclusion, our findings reveal two new PPIs between SOD1 and YWHAE or YWHAZ and their structural dependences, responses to redox status, mutual impacts on the enzyme function and protein degradation, and metabolic implications. Overall, our finding revealed a new unorthodox role of SOD1 and will provide novel perspectives and insights for diagnosing and treating diseases related to the protein.

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein-protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.

Lighting the way to host-directed immunotherapeutics

Developing broad-spectrum, host-directed antiviral therapeutics can be adapted to combat emerging viruses. In this issue of Cell Chemical Biology, Maarifi and colleagues implement a Nano luciferase reporter-based protein complementation assay to screen for small molecules and identify Gilteritinib, which enhances interferon induction and antagonizes virus replication.

Identifying enhancers of innate immune signaling as broad-spectrum antivirals active against emerging viruses

The increasingly frequent outbreaks of pathogenic viruses have underlined the urgent need to improve our arsenal of antivirals that can be deployed for future pandemics. Innate immunity is a powerful first line of defense against pathogens, and compounds that boost the innate response have high potential to act as broad-spectrum antivirals. Here, we harnessed localization-dependent protein-complementation assays (called Alpha Centauri) to measure the nuclear translocation of interferon regulatory factors (IRFs), thus providing a readout of innate immune activation following viral infection that is applicable to high-throughput screening of immunomodulatory molecules. As proof of concept, we screened a library of kinase inhibitors on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and identified Gilteritinib as a powerful enhancer of innate responses to viral infection. This immunostimulatory activity of Gilteritinib was found to be dependent on the AXL-IRF7 axis and results in a broad and potent antiviral activity against unrelated RNA viruses.

A synthetic switch based on orange carotenoid protein to control blue-green light responses in chloroplasts.

Synthetic biology approaches to engineer light-responsive systems are widely used, but their applications in plants are still limited due to the interference with endogenous photoreceptors and the intrinsic requirement of light for photosynthesis. Cyanobacteria possess a family of soluble carotenoid-associated proteins named orange carotenoid proteins (OCPs) that, when activated by blue–green light, undergo a reversible conformational change that enables the photoprotection mechanism that occurs on the phycobilisome. Exploiting this system, we developed a chloroplast-localized synthetic photoswitch based on a protein complementation assay where two nanoluciferase fragments were fused to separate polypeptides corresponding to the OCP2 domains. Since Arabidopsis (Arabidopsis thaliana) does not possess the prosthetic group needed for the assembly of the OCP2 complex, we first implemented the carotenoid biosynthetic pathway with a bacterial β-carotene ketolase enzyme (crtW) to generate keto-carotenoid-producing plants. The photoswitch was tested and characterized in Arabidopsis protoplasts and stably transformed plants with experiments aimed to uncover its regulation by a range of light intensities, wavelengths, and its conversion dynamics. Finally, we applied the OCP-based photoswitch to control transcriptional responses in chloroplasts in response to green light illumination by fusing the two OCP fragments with the plastidial SIGMA FACTOR 2 and bacteriophage T4 anti-sigma factor AsiA. This pioneering study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses upon exposure to specific light spectra.

Split-enzyme immunoassay to monitor EGFR-HER2 heterodimerization on cell surfaces

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. Statement of SignificanceWe describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts – two short peptide “tags” and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Expression, Purification and Crystallization of Asrij, A Novel Scaffold Transmembrane Protein.

Asrij/OCIAD1 is a scaffold transmembrane protein belonging to the Ovarian Carcinoma Immunoreactive Antigen Domain containing protein family. In Drosophila and mouse models, Asrij localizes at the endosomal and mitochondrial membrane and is shown to regulate the stemness of hematopoietic stem cells. Interaction of Asrij with ADP Ribosylation Factor 1 (Arf1) is shown to be crucial for hematopoietic niche function and prohemocyte maintenance. Here, we report the heterologous expression, standardization of detergents and purification methodologies for crystallization of Asrij/OCIAD1. To probe the activity of bacterially expressed Asrij, we developed a protein complementation assay and conclusively show that Asrij and Arf1 physically interact. Further, we find that sophorolipids improve the solubility and monodispersibility of Asrij. Hence, we propose that sophorolipids could be novel additives for stabilization of membrane proteins. To our knowledge, this is the first study detailing methodology for the production and crystallization of a heterologously expressed scaffold membrane protein and will be widely applicable to understand membrane protein structure and function.

Measuring the subcellular compartmentalization of viral infections by protein complementation assay

The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.

Proteomic Analysis Uncovers Measles Virus Protein C Interaction With p65–iASPP Protein Complex

AbstractViruses manipulate the central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two nonstructural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host-interacting proteins are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host-interacting proteins of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines, we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein interaction partners of MV-C were determined by applying protein complementation assay and the bioluminescence resonance energy transfer approach. As a result, we found that MV-C protein specifically interacts with p65–iASPP protein complex that controls both cell death and innate immunity pathways and evaluated the significance of these host factors on virus replication.

Split-Enzyme Immunoassay to Monitor EGFR-HER2 Heterodimerization on Cell Surfaces

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, energy-resonance transfer (ERT) assays and protein complementation assays (PCA), face significant limitations. ERT assays have a narrow working range of 10 to 50 A, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a novel assay platform to measure PPIs without modification of the proteins of interest and is functional at a greater working range than ERT assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition

SAT-LB124 Phosphatidylcholine Transfer Protein Interacts With PPARδ to Modulate Activity

Obesity is one of the largest public health crises in the USA. Obesity can be lethal due to the development of cardiovascular complications such as, hypertension, heart attack, and stroke. Recently, peroxisome proliferator-activated receptor δ (PPARδ), the least-characterized member of the PPAR family of nuclear receptors (NRs), has shown great promise in treating obesity and associated cardiovascular complications. Recent reports have shown that PPARδ activation is tuned, in part, through an interaction with fatty acid binding protein 5 (FABP5) in a polyunsaturated Fatty acids (PUFAs) dependent manner. This enhancement is thought to occur due to ligand transfer, however, FABP5 only binds a subset of reported PPARδ ligands. To find other candidate lipid transport proteins (LTPs), we performed a protein complementation assay (PCA) between LTPs and NRs. We uncovered a novel interaction between PPARδ and phosphatidylcholine transfer protein (PC-TP), sensitive to cellular nutrient levels. Preliminary data show that this interaction opposes canonical PPARδ signaling, leading to a decrease in PPARδ transactivation in cells and isolated mice livers. This led me to hypothesize that PC-TP senses nutrient status through membrane composition. Specific PC molecular species drive PC-TP translocation to inhibit PPARδ transactivation of genes. Utilizing our novel PCA assay I have shown that the interaction is modulated in part by cellular levels of methionine and choline, as cells cultured in media depleted of methionine and choline (MCD) show a decreased in the interaction. Using the same assay, I have shown a requirement of full length PPARδ for the interaction with PC-TP. This analysis will be complimented by mutagenesis and chemical perturbations aimed to alter PC-TP and PPARδ function. Additionally, I have assayed the in vivo relevance of PC-TP interaction with PPARδ using livers harvested from PCTP-/- and littermate control mice fed a variety of diets. Preliminary RNA-seq characterization, show interesting alterations in gene expression that suggest more complex regulation between PC-TP and PPARδ in vivo. MCD, which was shown to reduce the inhibitory interaction between PC-TP and PPARδ in vitro, seems to lead to an increased effect on PPARδ transactivation when PC-TP is depleted. Analysis comparing the effect of diet within each genotype shows a loss of differential PPARδ regulation for both CHEA and KEGG analysis when comparing WT to PCTP -/- mice supporting a role for PC-TP in differential PPARδ regulation caused by MCD diet. These studies will further the understanding of how lipid homeostasis is sensed and maintained through PPARδ by interactions with two separate LTPs.

X-ray Structure of the Human Karyopherin RanBP5, an Essential Factor for Influenza Polymerase Nuclear Trafficking

Here, we describe the crystal structures of two distinct isoforms of ligand-free human karyopherin RanBP5 and investigate its global propensity to interact with influenza A virus polymerase. Our results confirm the general architecture and mechanism of the IMB3 karyopherin-β subfamily whilst also highlighting differences with the yeast orthologue Kap121p. Moreover, our results provide insight into the structural flexibility of β-importins in the unbound state. Based on docking of a nuclear localisation sequence, point mutations were designed, which suppress influenza PA-PB1 subcomplex binding to RanBP5 in a binary protein complementation assay.

MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491

AbstractMutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.

In Vivo Protein Complementation Demonstrates Presynaptic α-Synuclein Oligomerization and Age-Dependent Accumulation of 8–16-mer Oligomer Species

Intracellular accumulation of α-synuclein (α-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson's disease (PD) and related α-synucleinopathies. Oligomerization and spreading of α-syn from neuron to neuron have been suggested as key events contributing to the progression of PD. To directly visualize and characterize α-syn oligomerization and spreading invivo, we generated two independentconditional transgenic mouse models based on α-syn protein complementation assays using neuron-specifically expressed split Gaussia luciferase or split Venus yellow fluorescent protein (YFP). These transgenic mice allow direct assessment of the quantity and subcellular distribution of α-syn oligomers invivo. Using these mouse models, we demonstrate an age-dependent accumulation of a specific subtype of α-syn oligomers. We provide invivo evidence that, although α-syn is found throughout neurons, α-syn oligomerization takes place at the presynapse. Furthermore, our mouse models provide strong evidence for a transsynaptic cell-to-cell transfer of de novo generated α-syn oligomers invivo.

The Interactome analysis of the Respiratory Syncytial Virus protein M2-1 suggests a new role in viral mRNA metabolism post-transcription

Human respiratory syncytial virus (RSV) is a globally prevalent negative-stranded RNA virus, which can cause life-threatening respiratory infections in young children, elderly people and immunocompromised patients. Its transcription termination factor M2-1 plays an essential role in viral transcription, but the mechanisms underpinning its function are still unclear. We investigated the cellular interactome of M2-1 using green fluorescent protein (GFP)-trap immunoprecipitation on RSV infected cells coupled with mass spectrometry analysis. We identified 137 potential cellular partners of M2-1, among which many proteins associated with mRNA metabolism, and particularly mRNA maturation, translation and stabilization. Among these, the cytoplasmic polyA-binding protein 1 (PABPC1), a candidate with a major role in both translation and mRNA stabilization, was confirmed to interact with M2-1 using protein complementation assay and specific immunoprecipitation. PABPC1 was also shown to colocalize with M2-1 from its accumulation in inclusion bodies associated granules (IBAGs) to its liberation in the cytoplasm. Altogether, these results strongly suggest that M2-1 interacts with viral mRNA and mRNA metabolism factors from transcription to translation, and imply that M2-1 may have an additional role in the fate of viral mRNA downstream of transcription.