scholarly journals New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1460
Author(s):  
Olga Dolnik ◽  
Gesche K. Gerresheim ◽  
Nadine Biedenkopf
Keyword(s):  
Inclusion Bodies ◽  
Rna Binding ◽  
Rna Virus ◽  
Virus Infections ◽  
Binding Domains ◽  
Protein Functions ◽  
Viral Inclusion ◽  
Dynamic Structures ◽  
And Function ◽  
Nucleoprotein N

Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane-less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to different protein functions. P keeps N soluble after expression to allow a concerted binding of N to the viral RNA. This results in the encapsidation of the viral genome by N, while P acts additionally as a cofactor for the viral polymerase, enabling viral transcription and replication. Here, we will review the formation and function of those viral inclusion bodies upon infection with NSVs with respect to their nature as biomolecular condensates.

scholarly journals tRNA 2′-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila

2020 ◽  
Vol 48 (4) ◽  
pp. 2050-2072 ◽  
Author(s):  
Margarita T Angelova ◽  
Dilyana G Dimitrova ◽  
Bruno Da Silva ◽  
Virginie Marchand ◽  
Caroline Jacquier ◽  
...  
Keyword(s):  
Small Rna ◽  
Rna Structure ◽  
Rna Silencing ◽  
Defense Mechanisms ◽  
Rna Virus ◽  
Virus Infections ◽  
Self Recognition ◽  
Silencing Pathways ◽  
Rna Pathways ◽  
And Function

Abstract 2′-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

scholarly journals Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

2005 ◽  
Vol 79 (15) ◽  
pp. 10023-10031 ◽  
Author(s):  
Jens Modrof ◽  
Kostas Lymperopoulos ◽  
Polly Roy
Keyword(s):  
Bluetongue Virus ◽  
Inclusion Bodies ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Nonstructural Protein 2 ◽  
The Matrix ◽  
Infected Cells ◽  
Viral Inclusion

ABSTRACT In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.

scholarly journals Formation and Function of Liquid-Like Viral Factories in Negative-Sense Single-Stranded RNA Virus Infections

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 126
Author(s):  
Justin M. Su ◽  
Maxwell Z. Wilson ◽  
Charles E. Samuel ◽  
Dzwokai Ma
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Host Responses ◽  
Future Research ◽  
Virus Infections ◽  
Infected Cells ◽  
And Function ◽  
Negative Sense ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) represents a major physiochemical principle to organize intracellular membrane-less structures. Studies with non-segmented negative-sense (NNS) RNA viruses have uncovered a key role of LLPS in the formation of viral inclusion bodies (IBs), sites of viral protein concentration in the cytoplasm of infected cells. These studies further reveal the structural and functional complexity of viral IB factories and provide a foundation for their future research. Herein, we review the literature leading to the discovery of LLPS-driven formation of IBs in NNS RNA virus-infected cells and the identification of viral scaffold components involved, and then outline important questions and challenges for IB assembly and disassembly. We discuss the functional implications of LLPS in the life cycle of NNS RNA viruses and host responses to infection. Finally, we speculate on the potential mechanisms underlying IB maturation, a phenomenon relevant to many human diseases.

scholarly journals tRNA 2’-O-methylation modulates small RNA silencing and life span in Drosophila

2019 ◽  
Author(s):  
Margarita T. Angelova ◽  
Dilyana G. Dimitrova ◽  
Bruno Da Silva ◽  
Virginie Marchand ◽  
Catherine Goyenvalle ◽  
...  
Keyword(s):  
Life Span ◽  
Small Rna ◽  
Rna Structure ◽  
Rna Silencing ◽  
Defense Mechanisms ◽  
Rna Virus ◽  
Virus Infections ◽  
Self Recognition ◽  
Rna Pathways ◽  
And Function

Abstract2’-O-methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two novel Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1, respectively. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while subsequently, CG5220 methylates position C32 in the same tRNAs and targets also additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as life span reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalysing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

scholarly journals Staufen2 mediated RNA recognition and localization requires combinatorial action of multiple domains

2018 ◽  
Author(s):  
Simone Heber ◽  
Imre Gáspár ◽  
Jan-Niklas Tants ◽  
Johannes Günther ◽  
Sandra M. Fernandez Moya ◽  
...  
Keyword(s):  
Rna Binding ◽  
Mrna Localization ◽  
Rna Recognition ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Physiological Relevance ◽  
And Function ◽  
Necessary And Sufficient ◽  
Multiple Domains ◽  
Mrna Binding

AbstractThroughout metazoans, Staufen (Stau) proteins are core factors of mRNA localization particles. They consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2) like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that only dsRBDs 3-4 are necessary and sufficient for mRNA binding. Here, we show that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient, all four dsRBDs recognize their target RNAs with high stability. Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization. In contrast, a rescue with mStau2 bearing RNA-binding mutations in dsRBD1-2 fails, confirming the physiological relevance of our findings. In summary, our data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau- family proteins of different species.

scholarly journals The devil is in the domain: understanding protein recognition of multiple RNA targets

2017 ◽  
Vol 45 (6) ◽  
pp. 1305-1311 ◽  
Author(s):  
Glen R. Gronland ◽  
Andres Ramos
Keyword(s):  
Rna Binding ◽  
Gene Expression Regulation ◽  
Rna Binding Proteins ◽  
Rna Regulation ◽  
Control Of Gene Expression ◽  
Binding Domains ◽  
Large Sets ◽  
Rna Targets ◽  
And Function ◽  
The Devil

RNA regulation provides a finely tuned and highly co-ordinated control of gene expression. Regulation is mediated by hundreds to thousands of multi-functional RNA-binding proteins which often interact with large sets of RNAs. In this brief review, we focus on a recent work that highlights how the proteins use multiple RNA-binding domains to interact selectively with the different RNA targets. Deconvoluting the molecular complexity of the RNA regulatory network is essential to understanding cell differentiation and function, and requires accurate models for protein–RNA recognition and protein target selectivity. We discuss that the structural and molecular understanding of the key determinant of recognition, together with the availability of methods to examine protein–RNA interactions at the transcriptome level, may provide an avenue to establish these models.

scholarly journals Structure of the Nucleoprotein Binding Domain of Mokola Virus Phosphoprotein

2009 ◽  
Vol 84 (2) ◽  
pp. 1089-1096 ◽  
Author(s):  
René Assenberg ◽  
Olivier Delmas ◽  
Jingshan Ren ◽  
Pierre-Olivier Vidalain ◽  
Anil Verma ◽  
...  
Keyword(s):  
Rna Binding ◽  
Crystal Packing ◽  
Rna Virus ◽  
Binding Domain ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Potential Binding Site ◽  
P Proteins ◽  
Nucleoprotein N ◽  
Mokola Virus

ABSTRACT Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli, which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae.

scholarly journals Structure and stability of theHuman respiratory syncytial virusM2–1RNA-binding core domain reveals a compact and cooperative folding unit

2017 ◽  
Vol 74 (1) ◽  
pp. 23-30 ◽  
Author(s):  
Ivana G. Molina ◽  
Inokentijs Josts ◽  
Yasser Almeida Hernandez ◽  
Sebastian Esperante ◽  
Mariano Salgueiro ◽  
...  
Keyword(s):  
Rna Binding ◽  
Tertiary Structure ◽  
Rna Virus ◽  
Binding Activity ◽  
Virus Family ◽  
Core Domain ◽  
New Family ◽  
Binding Core ◽  
And Function ◽  
Secondary And Tertiary Structure

Human syncytial respiratory virusis a nonsegmented negative-strand RNA virus with serious implications for respiratory disease in infants, and has recently been reclassified into a new family,Pneumoviridae. One of the main reasons for this classification is the unique presence of a transcriptional antiterminator, called M2–1. The puzzling mechanism of action of M2–1, which is a rarity among antiterminators in viruses and is part of the RNA polymerase complex, relies on dissecting the structure and function of this multidomain tetramer. The RNA-binding activity is located in a monomeric globular `core' domain, a high-resolution crystal structure of which is now presented. The structure reveals a compact domain which is superimposable on the full-length M2–1tetramer, with additional electron density for the C-terminal tail that was not observed in the previous models. Moreover, its folding stability was determined through chemical denaturation, which shows that the secondary and tertiary structure unfold concomitantly, which is indicative of a two-state equilibrium. These results constitute a further step in the understanding of this unique RNA-binding domain, for which there is no sequence or structural counterpart outside this virus family, in addition to its implications in transcription regulation and its likeliness as an antiviral target.

scholarly journals A Live-Cell Assay for the Detection of pre-microRNA-Protein Interactions

2020 ◽  
Author(s):  
Sydney L. Rosenblum ◽  
Daniel A. Lorenz ◽  
Amanda L. Garner
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Live Cell ◽  
Cell Detection ◽  
Binding Domains ◽  
Genome Wide ◽  
Rna Interaction ◽  
And Function ◽  
Specific Sequences

AbstractRecent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of both coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we have utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.

scholarly journals Human metapneumovirus P protein independently drives phase separation and recruits N protein to liquid-like inclusion bodies

2021 ◽  
Author(s):  
Kerri Beth Boggs ◽  
Nicolas Cifuentes-Munoz ◽  
Kearstin Edmonds ◽  
Farah El Najjar ◽  
Conny Ossandón ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nucleic Acid ◽  
Inclusion Bodies ◽  
Human Metapneumovirus ◽  
Liquid Droplets ◽  
N Protein ◽  
P Proteins ◽  
Dynamic Structures ◽  
Nucleoprotein N

Human metapneumovirus (HMPV) inclusion bodies (IBs) are dynamic structures required for efficient viral replication and transcription. The minimum components needed to form IB-like structures in cells are the nucleoprotein (N) and the tetrameric phosphoprotein (P). HMPV P binds to two versions of N protein in infected cells: C-terminal P residues interact with oligomeric, RNA-bound N (N-RNA), and N-terminal P residues interact with monomeric N (N0) to maintain a pool of protein to encapsidate new RNA. Recent work on other negative-strand viruses has suggested that IBs are liquid-like organelles formed via liquid-liquid phase separation (LLPS). Here, HMPV IBs in infected or transfected cells were shown to possess liquid organelle properties, such as fusion and fission. Recombinant versions of HMPV N and P proteins were purified to analyze the interactions required to drive LLPS in vitro. Purified HMPV P was shown to form liquid droplets in the absence of other protein binding partners, a novel finding compared to other viral systems. Removal of nucleic acid from purified P altered phase separation dynamics, suggesting that nucleic acid interactions also play a role in IB formation.  HMPV P also recruits monomeric N (N0-P) and N-RNA to IBs in vitro. These findings suggest that, in contrast to what has been reported for other viral systems, HMPV P acts as a scaffold protein to mediate multivalent interactions with monomeric and oligomeric HMPV N to promote phase separation of IBs.

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