scholarly journals A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity

Science ◽  
2020 ◽  
Vol 369 (6499) ◽  
pp. 54-59 ◽  
Author(s):  
Alexander J. Meeske ◽  
Ning Jia ◽  
Alice K. Cassel ◽  
Albina Kozlova ◽  
Jingqiu Liao ◽  
...  
Keyword(s):  
Single Dose ◽  
Structural Biology ◽  
Conformational Changes ◽  
Antiviral Response ◽  
Viral Rna ◽  
Multiple Infections ◽  
Dna Cleaving ◽  
Crispr System ◽  
Listeria Seeligeri ◽  
Target Rna

The CRISPR RNA (crRNA)–guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri. Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.

scholarly journals Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes

2020 ◽  
Vol 48 (20) ◽  
pp. 11664-11674 ◽  
Author(s):  
Ivana Duic ◽  
Hisashi Tadakuma ◽  
Yoshie Harada ◽  
Ryo Yamaue ◽  
Katashi Deguchi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Antiviral Response ◽  
Fiber Formation ◽  
Viral Rna ◽  
Active Conformation ◽  
Viral Dsrna ◽  
Fiber Assembly ◽  
Signaling Domain

Abstract Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

scholarly journals Role of conformational heterogeneity in ligand recognition by viral RNA molecules

2021 ◽  
Author(s):  
Lev Levintov ◽  
Harish Vashisth
Keyword(s):  
Conformational Changes ◽  
Ribonucleic Acid ◽  
Viral Rna ◽  
Ligand Recognition ◽  
Conformational Heterogeneity ◽  
Rna Molecules ◽  
Environmental Stimuli

Ribonucleic acid (RNA) molecules are known to undergo conformational changes in response to various environmental stimuli including temperature, pH, and ligands. In particular, viral RNA molecules are a key example...

scholarly journals Treatment of intestinal helminthiasis: mebendazole only or mebendazole-pyrantel pamoate?

2007 ◽  
Vol 47 (5) ◽  
pp. 216
Author(s):  
Wisman Dalimunthe ◽  
Charles Siregar ◽  
Munar Lubis ◽  
Syahril Pasaribu ◽  
Chairuddin P. Lubis
Keyword(s):  
Single Dose ◽  
Negative Impact ◽  
Reduction Rate ◽  
Multiple Infections ◽  
Cure Rate ◽  
School Students ◽  
Pyrantel Pamoate ◽  
Significant Difference ◽  
Chi Squared ◽  
Egg Reduction Rate

Background Although intestinal helminthiasis causes highmorbidity and has a negative impact on children’s growth anddevelopment, the efficacy of antihelmintics for multiplehelminthiasis in mass treatment is still doubtful.Objective To compare the efficacy of single dose mebendazoleand a combination of pyrantel pamoate and mebendazole for thetreatment of multiple infections due to Ascaris lumbricoides,hookworm, and Trichuris trichiura.Methods Subjects were elementary school students in Suka Village,Tiga Panah subdistrict, North Sumatera. They were randomizedto either receive mebendazole (M Group) or mebendazole-pyrantel pamoate group (MP Group). Stool examinations wereperfomed on each subjects on day 7, 14, 21, and 28 after treatment.Analyses were perfomed by using chi-squared and Mann-WhitneyU tests.Results The prevalence of intestinal helminthiasis was 95.4%. T.trichiura (88.7%) was the most common cause of infection followedby A. lumbricoides (79.5%), and hookworm (3.1%). Two hundredthirty nine (76.8%) children had multiple infections. Althoughthe egg reduction rate of intestinal helminthiasis in thecombination group was faster than that of the mebendazole group,there was no significant difference in the cure rate of both groups.Conclusion A single dose of mebendazole is preferred for masstreatment of multiple intestinal helminthiasis infections.

scholarly journals Studying Conformational Changes of the Yersinia Type-III-Secretion Effector YopO in Solution by Integrative Structural Biology

Structure ◽  
2019 ◽  
Vol 27 (9) ◽  
pp. 1416-1426.e3 ◽  
Author(s):  
Martin F. Peter ◽  
Anne T. Tuukkanen ◽  
Caspar A. Heubach ◽  
Alexander Selsam ◽  
Fraser G. Duthie ◽  
...  
Keyword(s):  
Structural Biology ◽  
Conformational Changes ◽  
Type Iii Secretion ◽  
Type Iii ◽  
Integrative Structural Biology ◽  
Type Iii Secretion Effector

scholarly journals Complementary Mutations in the N and L Proteins for Restoration of Viral RNA Synthesis

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Weike Li ◽  
Ryan H. Gumpper ◽  
Yusuf Uddin ◽  
Ingeborg Schmidt-Krey ◽  
Ming Luo
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Rna Synthesis ◽  
Large Subunit ◽  
Viral Rna ◽  
Structural Motif ◽  
General Mechanism ◽  
Rna Dependent Rna Polymerase ◽  
N Protein ◽  
Rna Genome

ABSTRACTDuring viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif—Glu169, Phe171, and Leu174—to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid.IMPORTANCEDuring viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis.

scholarly journals Targeting RNA structures in diseases with small molecules

2020 ◽  
Vol 64 (6) ◽  
pp. 955-966 ◽  
Author(s):  
Yanqiu Shao ◽  
Qiangfeng Cliff Zhang
Keyword(s):  
Gene Expression ◽  
Molecular Biology ◽  
Small Molecules ◽  
Structural Biology ◽  
Rna Structure ◽  
Therapeutic Potential ◽  
Rna Structures ◽  
Gene Expression And Regulation ◽  
Cellular Processes ◽  
Target Rna

Abstract RNA is crucial for gene expression and regulation. Recent advances in understanding of RNA biochemistry, structure and molecular biology have revealed the importance of RNA structure in cellular processes and diseases. Various approaches to discovering drug-like small molecules that target RNA structure have been developed. This review provides a brief introduction to RNA structural biology and how RNA structures function as disease regulators. We summarize approaches to targeting RNA with small molecules and highlight their advantages, shortcomings and therapeutic potential.

scholarly journals Discerning best practices in XFEL-based biological crystallography – standards for nonstandard experiments

IUCrJ ◽  
2021 ◽  
Vol 8 (4) ◽  
pp. 532-543
Author(s):  
Alexander Gorel ◽  
Ilme Schlichting ◽  
Thomas R. M. Barends
Keyword(s):  
Best Practices ◽  
Structural Biology ◽  
Conformational Changes ◽  
Critical Evaluation ◽  
Quality Standards ◽  
Free Electron Lasers ◽  
High Resolution Data ◽  
X Ray ◽  
Serial Femtosecond Crystallography ◽  
Resolution Data

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a novel tool in structural biology. In contrast to conventional crystallography, SFX relies on merging partial intensities acquired with X-ray beams of often randomly fluctuating properties from a very large number of still diffraction images of generally randomly oriented microcrystals. For this reason, and possibly due to limitations of the still evolving data-analysis programs, XFEL-derived SFX data are typically of a lower quality than `standard' crystallographic data. In contrast with this, the studies performed at XFELs often aim to investigate issues that require precise high-resolution data, for example to determine structures of intermediates at low occupancy, which often display very small conformational changes. This is a potentially dangerous combination and underscores the need for a critical evaluation of procedures including data-quality standards in XFEL-based structural biology. Here, such concerns are addressed.

scholarly journals Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Ville Hoikkala ◽  
Janne Ravantti ◽  
César Díez-Villaseñor ◽  
Marja Tiirola ◽  
Rachel A. Conrad ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Flavobacterium Columnare ◽  
Content Type ◽  
Immune Systems ◽  
Dna Cleaving ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Rna Targeting ◽  
Microbial Strains ◽  
Target Rna

ABSTRACT CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules. IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader’s genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system “borrows” acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.

scholarly journals MELD-DNA: A new tool for capturing protein-DNA binding

2021 ◽  
Author(s):  
Antonio Bauza ◽  
Alberto Perez
Keyword(s):  
Structural Biology ◽  
Conformational Changes ◽  
Dna Sequences ◽  
Structure Prediction ◽  
Binding Mode ◽  
Binding Modes ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Multiple Binding ◽  
New Software Development

Herein we present MELD-DNA, a novel computational approach to address the problem of protein-DNA structure prediction. This method addresses well-known issues hampering current computational approaches to bridge the gap between structural and sequence knowledge, such as large conformational changes in DNA and highly charged electrostatic interaction during binding. MELD- DNA is able to: i) sample multiple binding modes, ii) identify the preferred binding mode from the ensembles, and iii) provide qualitative binding preferences between DNA sequences. We expect the results presented herein will have impact in the field of biophysics (through new software development), structural biology (by complementing DNA structural databases) and supramolecular chemistry (by bringing new insights into protein-DNA interactions).

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