scholarly journals Supramolecular cylinders target bulge structures in the 5’ UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication

2021 ◽  
Author(s):  
Lazaros Melidis ◽  
Harriet J. Hill ◽  
Nicholas J. Coltman ◽  
Scott P. Davies ◽  
Kinga Winczura ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Binding ◽  
Antiviral Agents ◽  
3D Models ◽  
Stem Loop ◽  
Dynamic Structures ◽  
Dynamics Simulations

AbstractThe untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with Molecular Dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5’ UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in the stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel antiviral agents.

scholarly journals Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1517
Author(s):  
Rebecca S. Brown ◽  
Lisa Kim ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Rna Structure ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Specific Binding ◽  
Genomic Rna ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Labeled Rna

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

scholarly journals Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

2021 ◽  
Author(s):  
Christine Roden ◽  
Yifan Dai ◽  
Ian Seim ◽  
Myungwoon Lee ◽  
Rachel Sealfon ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Genome Packaging ◽  
Rna Motifs ◽  
Double Stranded Rna ◽  
N Protein ◽  
Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

Viral RNA structural equilibria and their interactions with host proteins

2019 ◽  
Author(s):  
◽  
Samantha Elizabeth Brady
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Primer Binding Site ◽  
In Vitro Techniques

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.

scholarly journals Dissecting the Fine Details of Assembly of aT = 3 Phage Capsid

2005 ◽  
Vol 6 (2) ◽  
pp. 119-125 ◽  
Author(s):  
P. G. Stockley ◽  
A. E. Ashcroft ◽  
S. Francese ◽  
G. S. Thompson ◽  
N. A. Ranson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protein Complexes ◽  
Model Systems ◽  
Replicase Gene ◽  
Nmr Studies ◽  
Partial Explanation ◽  
Stem Loop ◽  
Assembly Pathway

The RNA bacteriophages represent ideal model systems in which to probe the detailed assembly pathway for the formation of aT = 3 quasi-equivalent capsid. For MS2, the assembly reaction can be probedin vitrousing acid disassembled coat protein subunits and a short (19 nt) RNA stem-loop that acts as the translational operator of the replicase gene and leads to sequence-specific sequestration and packaging of the cognate phage RNAin vivo. Reassembly reactions can be initiated by mixing these components at neutral pH. The molecular basis of the sequence-specific RNA–protein interaction is now well understood. Recent NMR studies on the protein demonstrate extensive mobility in the loops of the polypeptide that alter their conformations to form the quasi-equivalent conformers of the final capsid. It seems reasonable to assume that RNA binding results in reduction of this flexibility. However, mass spectrometry suggests that these RNA–protein complexes may only provide one type of quasi-equivalent capsid building block competent to form five-fold axes but not the full shell. Work with longer RNAs suggests that the RNA may actively template the assembly pathway providing a partial explanation of how conformers are selected in the growing shell.

scholarly journals HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

2015 ◽  
Vol 90 (5) ◽  
pp. 2332-2344 ◽  
Author(s):  
Rong Yu ◽  
Darong Yang ◽  
Shaohua Lei ◽  
Xiaohong Wang ◽  
Xianghe Meng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Untranslated Region ◽  
Rna Binding ◽  
High Mobility ◽  
Hcv Rna ◽  
Functional Domain ◽  
Hmgb1 Protein ◽  
Stem Loop

ABSTRACTHigh-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome.IMPORTANCEHepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is warranted to elucidate the specific actions of HMGB1 in HCV pathogenesis.

scholarly journals Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein

2005 ◽  
Vol 393 (1) ◽  
pp. 373-379 ◽  
Author(s):  
Akio Kanai ◽  
Asako Sato ◽  
Jun Imoto ◽  
Masaru Tomita
Keyword(s):  
Thymidylate Synthase ◽  
Rna Binding ◽  
Pyrococcus Furiosus ◽  
In Vitro Translation ◽  
Luciferase Reporter ◽  
Amino Acid Sequence Similarity ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

Using a stem–loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem–loop structure at a high temperature (75 °C) with an apparent dissociation constant of 0.6 μM. A similar stem–loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem–loop structure. In vitro translation analysis using chimaeric constructs containing the stem–loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem–loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine–Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.

scholarly journals Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

2008 ◽  
Vol 83 (2) ◽  
pp. 993-1008 ◽  
Author(s):  
María F. Lodeiro ◽  
Claudia V. Filomatori ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Stem Loop ◽  
Denv Serotypes ◽  
Functional Studies ◽  
Promoter Recognition

ABSTRACT The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG region (5′UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito- and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5′UAR, was necessary for efficient RNA synthesis using the viral 3′UTR as template.

scholarly journals RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein

2021 ◽  
Vol 22 (16) ◽  
pp. 9103
Author(s):  
Julita Gumna ◽  
Angelika Andrzejewska-Romanowska ◽  
David J. Garfinkel ◽  
Katarzyna Pachulska-Wieczorek
Keyword(s):  
Rna Structure ◽  
Rna Binding ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Mobility Shift ◽  
Gag Protein ◽  
Rna Dimerization ◽  
Electrophoretic Mobility Shift Assays

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

scholarly journals Accurate Detection of RNA Stem-Loops in Structurome Data Reveals Widespread Association with Protein Binding Sites

2021 ◽  
Author(s):  
Pierce Radecki ◽  
Rahul Uppuluri ◽  
Kaustubh Deshpande ◽  
Sharon Aviran
Keyword(s):  
Rna Structure ◽  
Nearest Neighbor ◽  
Rna Binding ◽  
Learning Algorithm ◽  
Accurate Method ◽  
Stem Loop ◽  
Rna Molecules ◽  
Computational Overhead ◽  
Local Sequence ◽  
Functional Relevance

ABSTRACTRNA molecules are known to fold into specific structures which often play a central role in their functions and regulation. In silico folding of RNA transcripts, especially when assisted with structure profiling (SP) data, is capable of accurately elucidating relevant structural conformations. However, such methods scale poorly to the swaths of SP data generated by transcriptome-wide experiments, which are becoming more commonplace and advancing our understanding of RNA structure and its regulation at global and local levels. This has created a need for tools capable of rapidly deriving structural assessments from SP data in a scalable manner. One such tool we previously introduced that aims to process such data is patteRNA, a statistical learning algorithm capable of rapidly mining big SP datasets for structural elements. Here, we present a reformulation of patteRNA’s pattern recognition scheme that sees significantly improved precision without major compromises to computational overhead. Specifically, we developed a data-driven logistic classifier which interprets patteRNA’s statistical characterizations of SP data in addition to local sequence properties as measured with a nearest neighbor thermodynamic model. Application of the classifier to human structurome data reveals a marked association between detected stem-loops and RNA binding protein (RBP) footprints. The results of our application demonstrate that upwards of 30% of RBP footprints occur within loops of stable stem-loop elements. Overall, our work arrives at a rapid and accurate method for automatically detecting families of RNA structure motifs and demonstrates the functional relevance of identifying them transcriptome-wide.

scholarly journals In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0246319
Author(s):  
Raphael J. Eberle ◽  
Danilo S. Olivier ◽  
Carolina C. Pacca ◽  
Clarita M. S. Avilla ◽  
Mauricio L. Nogueira ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Chikungunya Virus ◽  
In Vitro Study ◽  
3D Models ◽  
Binding Pocket ◽  
Starting Point ◽  
Low Cytotoxicity ◽  
Inhibitory Potential ◽  
Dynamics Simulations

The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.

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