RNA binding property and RNA chaperone activity of dengue virus core protein and other viral RNA-interacting proteins.
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ABSTRACT:
SUBMITTER: Pong WL
PROVIDER: S-EPMC7164067 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:UnlabelledDengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.ImportanceDengue virus is the most prevalent cause of arthropod-borne infection worldwide. Viral RNA molecules physically interact with cellular RNA-binding proteins (RBPs) throughout the course of infection; the identification of such interactions will lead to the elucidation of the molecular mechanisms of virus replication. Until now, the identification of host proteins bound to dengue viral RNA has been accomplished using in vitro strategies. Here, we used a method for the specific purification of dengue viral ribonucleoprotein (RNP) complexes from infected cells and subsequently identified the associated proteins by mass spectrometry. We then validated a functional role for the majority of these proteins in mediating efficient virus replication. This approach has broad relevance to virology and RNA biology, as it could theoretically be used to purify any viral RNP complex of interest.
| S-EPMC4725007 | biostudies-literature
Project description:Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no effective treatment is available. DENV relies heavily on the host cellular machinery for productive infection. Here, we show that the scaffold protein RACK1, which is part of the DENV replication complex, mediates infection by binding to the 40S ribosomal subunit. Mass spectrometry analysis of RACK1 partners coupled to an RNA interference screen-identified Vigilin and SERBP1 as DENV host-dependency factors. Both are RNA-binding proteins that interact with the DENV genome. Genetic ablation of Vigilin or SERBP1 rendered cells poorly susceptible to DENV, as well as related flaviviruses, by hampering the translation and replication steps. Finally, we established that a Vigilin or SERBP1 mutant lacking RACK1 binding but still interacting with the viral RNA is unable to mediate DENV infection. We propose that RACK1 recruits Vigilin and SERBP1, linking the DENV genome to the translation machinery for efficient infection. IMPORTANCE We recently identified the scaffolding RACK1 protein as an important host-dependency factor for dengue virus (DENV), a positive-stranded RNA virus responsible for the most prevalent mosquito-borne viral disease worldwide. Here, we have performed the first RACK1 interactome in human cells and identified Vigilin and SERBP1 as DENV host-dependency factors. Both are RNA-binding proteins that interact with the DENV RNA to regulate viral replication. Importantly, Vigilin and SERBP1 interact with RACK1 and the DENV viral RNA (vRNA) to mediate viral replication. Overall, our results suggest that RACK1 acts as a binding platform at the surface of the 40S ribosomal subunit to recruit Vigilin and SERBP1, which may therefore function as linkers between the viral RNA and the translation machinery to facilitate infection.
| S-EPMC9006918 | biostudies-literature
Project description:BackgroundThere are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication.Methodology/principal findingsSeventy-nine novel RNA binding proteins for dengue virus (DENV) were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated.Conclusions/significanceThe method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps cellular proteins for efficient amplification.
| S-EPMC4996428 | biostudies-literature
Project description:Arrays of >5,000 Saccharomyces cerevisiae proteins were screened to identify proteins that can preferentially bind a small RNA hairpin that contains a clamped adenine motif (CAM). A CAM is required for the replication of Brome Mosaic Virus (BMV), a plant-infecting RNA virus that can replicate in S. cerevisiae. Several hits were selected for further characterization in Nicotiana benthamiana. Pseudouridine Synthase 4 (Pus4) and the Actin Patch Protein 1 (App1) modestly reduced BMV genomic plus-strand RNA accumulation, but dramatically inhibited BMV systemic spread in plants. Pus4 also prevented the encapsidation of a BMV RNA in plants and the reassembly of BMV virions in vitro. These results demonstrate the feasibility of using proteome arrays to identify specific RNA-binding proteins for antiviral activities. Furthermore, the effects of Pus4 suggest that the CAM-containing RNA motif provides a regulatory link between RNA replication and encapsidation.
| S-EPMC1805585 | biostudies-literature
Project description:Dengue viruses (DENV) are expanding global pathogens that are transmitted through the bite of mosquitoes, mostly Aedes aegypti. As RNA viruses, DENV rely on RNA-binding proteins (RBPs) to complete their life cycle. Alternatively, RBPs can act as restriction factors that prevent DENV multiplication. While the importance of RBPs is well-supported in humans, there is a dearth of information about their influence on DENV transmission by mosquitoes. Such knowledge could be harnessed to design novel, effective interventions against DENV. Here, we successfully adapted RNA-affinity chromatography coupled with mass spectrometry-a technique initially developed in mammalian cells-to identify RBPs in Ae. aegypti cells. We identified fourteen RBPs interacting with DENV serotype 2 3'UTR, which is involved in the viral multiplication and produces subgenomic flaviviral RNA (sfRNA). We validated the RNA affinity results for two RBPs by confirming that AePur binds the 3'UTR, whereas AeStaufen interacts with both 3'UTR and sfRNA. Using in vivo functional evaluation, we determined that RBPs like AeRan, AeExoRNase, and AeRNase have pro-viral functions, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes. Furthermore, we showed that human and mosquito Pur homologs have a shared affinity to DENV2 RNA, although the anti-viral effect is specific to the mosquito protein. Importantly, we revealed that AeStaufen mediates a reduction of gRNA and sfRNA copies in several mosquito tissues, including the salivary glands and that AeStaufen-mediated sfRNA reduction diminishes the concentration of transmission-enhancing sfRNA in saliva, thereby revealing AeStaufen's role in DENV transmission. By characterizing the first RBPs that associate with DENV2 3'UTR in mosquitoes, our study unravels new pro- and anti-viral targets for the design of novel therapeutic interventions as well as provides foundation for studying the role of RBPs in virus-vector interactions.
| S-EPMC9531803 | biostudies-literature
Project description:The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.
| S-EPMC419467 | biostudies-literature
Project description:Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.
| S-EPMC3192115 | biostudies-literature
Project description:Dengue virus (DENV), a re-emerging virus transmitted by Aedes mosquitoes, causes severe pathogenesis in humans. No effective treatment is available against this virus. We recently identified the scaffold protein RACK1 as a component of the DENV replication complex, a macromolecular complex essential for viral genome amplification. Here, we show that RACK1 is important for DENV infection. RACK1 mediates DENV replication through binding to the 40S ribosomal subunit. Mass spectrometry analysis of RACK1 partners coupled to a loss-of-function screen identified the RNA binding proteins Vigilin and SERBP1 as DENV host dependency factors. Vigilin and SERBP1 interact with DENV viral RNA (vRNA), forming a ternary complex with RACK1 to mediate viral replication. Overall, our results indicate that RACK1 recruits Vigilin and SERBP1, linking the DENV vRNA to the translation machinery for optimal translation and replication.
2022-02-04 | PXD030765 | Pride
Project description:ObjectiveGlucagon in mammals and its homolog (adipokinetic hormone [AKH] in Drosophila melanogaster) are peptide hormones which regulate lipid metabolism by breaking down triglycerides. Although regulatory mechanisms of glucagon and AKH expression have been widely studied, post-transcriptional gene expression of glucagon has not been investigated thoroughly. In this study, we aimed to profile proteins binding with Gcg messenger RNA (mRNA) in mouse and Akh mRNA in Drosophila.MethodsDrosophila Schneider 2 (S2) and mouse 3T3-L1 cell lysates were utilized for affinity pull down of Akh and Gcg mRNA respectively using biotinylated anti-sense DNA oligoes against target mRNAs. Mass spectrometry and computational network analysis revealed mRNA-interacting proteins residing in functional proximity.ResultsWe observed that 1) 91 proteins interact with Akh mRNA from S2 cell lysates, 2) 34 proteins interact with Gcg mRNA from 3T3-L1 cell lysates. 3) Akh mRNA interactome revealed clusters of ribosomes and known RNA-binding proteins (RBPs). 4) Gcg mRNA interactome revealed mRNA-binding proteins including Plekha7, zinc finger protein, carboxylase, lipase, histone proteins and a cytochrome, Cyp2c44. 5) Levels of Gcg mRNA and its interacting proteins are elevated in skeletal muscles isolated from old mice compared to ones from young mice.ConclusionAkh mRNA in S2 cells are under active translation in a complex of RBPs and ribosomes. Gcg mRNA in mouse precursor adipocyte is in a condition distinct from Akh mRNA due to biochemical interactions with a subset of RBPs and histones. We anticipate that our study contributes to investigating regulatory mechanisms of Gcg and Akh mRNA decay, translation, and localization.
| S-EPMC8792818 | biostudies-literature
Project description:Up to now less than a handful of viral cholesterol-binding proteins have been characterized, in HIV, influenza virus and Semliki Forest virus. These are proteins with roles in virus entry or morphogenesis. In the case of the HIV fusion protein gp41 cholesterol binding is attributed to a cholesterol recognition consensus (CRAC) motif in a flexible domain of the ectodomain preceding the trans-membrane segment. This specific CRAC sequence mediates gp41 binding to a cholesterol affinity column. Mutations in this motif arrest virus fusion at the hemifusion stage and modify the ability of the isolated CRAC peptide to induce segregation of cholesterol in artificial membranes.Influenza A virus M2 protein co-purifies with cholesterol. Its proton translocation activity, responsible for virus uncoating, is not cholesterol-dependent, and the transmembrane channel appears too short for integral raft insertion. Cholesterol binding may be mediated by CRAC motifs in the flexible post-TM domain, which harbours three determinants of binding to membrane rafts. Mutation of the CRAC motif of the WSN strain attenuates virulence for mice. Its affinity to the raft-non-raft interface is predicted to target M2 protein to the periphery of lipid raft microdomains, the sites of virus assembly. Its influence on the morphology of budding virus implicates M2 as factor in virus fission at the raft boundary. Moreover, M2 is an essential factor in sorting the segmented genome into virus particles, indicating that M2 also has a role in priming the outgrowth of virus buds.SFV E1 protein is the first viral type-II fusion protein demonstrated to directly bind cholesterol when the fusion peptide loop locks into the target membrane. Cholesterol binding is modulated by another, proximal loop, which is also important during virus budding and as a host range determinant, as shown by mutational studies.
| S-EPMC7176229 | biostudies-literature