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1.
Pestic Biochem Physiol ; 179: 104966, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34802516

RESUMO

Pesticide resistance in spider mites drives the development of acaricides with novel mode of action, which could benefit from RNAi as a screening tool in search of new molecular targets. RNAi via oral delivery of dsRNA has been frequently reported in spider mites, but injection of dsRNA is rarely reported. We compare here the efficiency of oral delivery versus injection of dsRNA in female adult mites. When comparing silencing efficiency, oral delivery of dsRNAs silenced 40.6 ± 8.9% of CPR, 63.8 ± 6.9% of CHMP2A, and 37.7 ± 5.7% of CHMP3 genes. Similar silencing efficiencies were found for injection (48.6 ± 3.7% of CPR, 70.2 ± 4.1% of CHMP2A, 59.8 ± 2.2% of CHMP3), but with much lower quantities of dsRNAs. Oral delivery of dsRNA failed to silence the expression of the CHMP4B gene, but this could be accomplished by injection of dsRNA (23.1 ± 1.0%). When scoring the phenotypic effects of silencing, both oral delivery and injection of CHMP2A- and CHMP3-dsRNA influenced the locomotion speed of mites significantly. For CPR, silencing could only be accomplished by dsRNA injection, not by feeding. CPR silencing significantly impacted the toxicity of a typical acaricide, pyridaben, as the susceptibility of mites raised 2.75-fold. Last, injection of Eya-dsRNA in adults produced transgenerational phenotypic effects on 3.59% of offspring, as quantified by an observed deviation in eye development, while oral delivery of Eya-dsRNA did not. In conclusion, injection of dsRNA is superior to oral delivery in silencing the expression of the selected genes in this study and could be considered the method of choice to study gene function in reverse genetic approaches.


Assuntos
Acaricidas , Ácaros , Tetranychidae , Animais , Ácaros/genética , Interferência de RNA , RNA de Cadeia Dupla/genética
2.
Planta ; 254(6): 121, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34779907

RESUMO

MAIN CONCLUSION: Host-derived suppression of nematode essential genes decreases reproduction of Meloidogyne incognita in cotton. Root-knot nematodes (RKN) represent one of the most damaging plant-parasitic nematode genera worldwide. RNAi-mediated suppression of essential nematode genes provides a novel biotechnological strategy for the development of sustainable pest-control methods. Here, we used a Host Induced Gene Silencing (HIGS) approach by stacking dsRNA sequences into a T-DNA construct to target three essential RKN genes: cysteine protease (Mi-cpl), isocitrate lyase (Mi-icl), and splicing factor (Mi-sf), called dsMinc1, driven by the pUceS8.3 constitutive soybean promoter. Transgenic dsMinc1-T4 plants infected with Meloidogyne incognita showed a significant reduction in gall formation (57-64%) and egg masses production (58-67%), as well as in the estimated reproduction factor (60-78%), compared with the susceptible non-transgenic cultivar. Galls of the RNAi lines are smaller than the wild-type (WT) plants, whose root systems exhibited multiple well-developed root swellings. Transcript levels of the three RKN-targeted genes decreased 13- to 40-fold in nematodes from transgenic cotton galls, compared with those from control WT galls. Finally, the development of non-feeding males in transgenic plants was 2-6 times higher than in WT plants, indicating a stressful environment for nematode development after RKN gene silencing. Data strongly support that HIGS of essential RKN genes is an effective strategy to improve cotton plant tolerance. This study presents the first application of dsRNA sequences to target multiple genes to promote M. incognita tolerance in cotton without phenotypic penalty in transgenic plants.


Assuntos
Gossypium , Tylenchoidea , Animais , Gossypium/genética , Doenças das Plantas/genética , Plantas Geneticamente Modificadas/genética , RNA de Cadeia Dupla , Tylenchoidea/genética
3.
PLoS One ; 16(10): e0256070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34653190

RESUMO

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/metabolismo , Haemophilus influenzae/metabolismo , Heme/metabolismo , RNA de Cadeia Dupla/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Transporte/genética , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Otite Média/microbiologia , Otite Média/patologia , Conformação Proteica , Transporte Proteico/fisiologia , RNA de Cadeia Dupla/genética , Motivos de Ligação ao RNA/genética , Fatores de Virulência/metabolismo
4.
J Gen Virol ; 102(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34623234

RESUMO

In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.


Assuntos
Proteínas Argonauta/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Ribonuclease III/metabolismo , Tabaco/virologia , Tombusviridae/fisiologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Tabaco/genética , Tabaco/metabolismo , Tombusviridae/genética
5.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502076

RESUMO

The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL-CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated tumors in the case of RFA.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Linfócitos do Interstício Tumoral/efeitos dos fármacos , RNA de Cadeia Dupla/uso terapêutico , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/cirurgia , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Colo/cirurgia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/radioterapia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ablação por Radiofrequência , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
6.
J Insect Sci ; 21(5)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34581410

RESUMO

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.


Assuntos
Interferência de RNA , RNA de Cadeia Dupla/farmacologia , Spodoptera , Animais , Bactérias/genética , Quitinases/efeitos dos fármacos , Quitinases/genética , Fertilidade/efeitos dos fármacos , Genes de Insetos/efeitos dos fármacos , Imunoglobulinas/efeitos dos fármacos , Imunoglobulinas/genética , Proteínas de Insetos/efeitos dos fármacos , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/fisiologia , Controle Biológico de Vetores , Pupa/efeitos dos fármacos , Pupa/genética , Pupa/fisiologia , Reprodução/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Spodoptera/fisiologia
7.
Biochemistry (Mosc) ; 86(8): 1025-1040, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488578

RESUMO

The review discusses differences between the specific protein interactions with single- and double-stranded RNA molecules using the data on the structure of RNA-protein complexes. Proteins interacting with the single-stranded RNAs form contacts with RNA bases, which ensures recognition of specific nucleotide sequences. Formation of such contacts with the double-stranded RNAs is hindered, so that the proteins recognize unique conformations of the RNA spatial structure and interact mainly with the RNA sugar-phosphate backbone.


Assuntos
RNA de Cadeia Dupla/química , RNA/química , Motivos de Aminoácidos , Sequência de Bases , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Dedos de Zinco
8.
Nucleic Acids Res ; 49(18): 10604-10617, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34520542

RESUMO

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.


Assuntos
Algoritmos , RNA de Cadeia Dupla/química , RNA Mensageiro/química , RNA Viral/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Pareamento de Bases , Sequência de Bases , COVID-19/prevenção & controle , Humanos , Hidrólise , Estabilidade de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Viral/genética , RNA Viral/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Termodinâmica
9.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575949

RESUMO

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.


Assuntos
Oligodesoxirribonucleotídeos/genética , RNA de Cadeia Dupla/antagonistas & inibidores , RNA Interferente Pequeno/genética , Ribonucleases/genética , Guanidina/química , Humanos , Oligodesoxirribonucleotídeos/antagonistas & inibidores , Oligodesoxirribonucleotídeos/farmacologia , Interferência de RNA , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genética , Ribonucleases/química , Termodinâmica
10.
Arch Insect Biochem Physiol ; 108(3): e21840, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34569086

RESUMO

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a destructive pest which is famous for its resistance to a variety of insecticides. RNA interference is a posttranscriptional gene silencing mechanism that has become a popular tool to control insect pests, triggered by double-stranded RNAs (dsRNAs). The effect of ingestion and injection delivery methods of dsRNA related to some protease genes including Trypsin (Ha-TRY39 and Ha-TRY96), Chymotrypsin (Ha-CHY), and Cathepsin L (Ha-CAT) on growth and development of H. armigera was investigated in this study. All protease genes encoded full ORFs and were expressed in all H. armigera larvae stages and tissues. In both injection and feeding bioassays, Ha-RNAi CHY's performance outperformed that of other protease genes. CHY enzyme activity in the midgut of larvae was significantly reduced after treatment with ds-HaCHY. Oral administration of ds-CHY also resulted in significant mortality of H. armigera larvae. However, because of the high RNase activity in the midgut lumen of lepidoptera, a large amount of dsRNA was needed to effectively kill instars of H. armigera. To reduce dsRNA degradation, bacterial expression and dsRNA formulation were used. After oral administration, it was toxic to H. armigera larvae. Before oral administration, bacterial cells were sonicated to increase dsRNA release. The RNA interference efficiency of sonicated bacteria was significantly increased, resulting in higher larval mortality when administered orally. All of these findings point to Ha-CHY as a new candidate for developing an effective dsRNA-based pesticide for H. armigera control.


Assuntos
Mariposas , Peptídeo Hidrolases , RNA de Cadeia Dupla/farmacologia , Animais , Bactérias/genética , Catepsinas/efeitos dos fármacos , Catepsinas/genética , Quimotripsina/efeitos dos fármacos , Quimotripsina/genética , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Mortalidade , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Organismos Geneticamente Modificados , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/genética , Controle de Pragas/métodos , Interferência de RNA , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/metabolismo , Tripsina/efeitos dos fármacos , Tripsina/genética
11.
Arch Virol ; 166(11): 3229-3232, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34524536

RESUMO

The complete genome sequence of a double-stranded RNA (dsRNA) virus, Rhizoctonia solani dsRNA virus 11 (RsRV11), isolated from Rhizoctonia solani AG-1 IA strain 9-11 was determined. The RsRV11 genome is 9,555 bp in length and contains three conserved domains: structural maintenance of chromosomes (SMC) superfamily, phosphoribulokinase (PRK), and RNA-dependent RNA polymerase (RdRp). The RsRV11 genome has two non-overlapping open reading frames (ORFs). ORF1 is predicted to encode a 204.12-kDa protein that shares low but significant amino acid sequence similarity with a putative protein encoded by Rhizoctonia solani RNA virus HN008 (RsRV-HN008). ORF2 potentially encodes a 132.41-kDa protein that contains the conserved domain of the RdRp. Phylogenetic analysis indicated that RsRV11 clustered with RsRV-HN008 in a separate clade from other virus families. This implies that RsRV11 and RsRV-HN008 should be included in a new mycovirus taxon close to the family Megabirnaviridae and that RsRV11 is a new mycovirus.


Assuntos
Micovírus/genética , Genoma Viral , Filogenia , Rhizoctonia/virologia , China , Micovírus/isolamento & purificação , Fases de Leitura Aberta , RNA de Cadeia Dupla , Rhizoctonia/isolamento & purificação , Proteínas Virais/genética , Zea mays/microbiologia
12.
Arch Virol ; 166(11): 3233-3237, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34535823

RESUMO

The entomopathogenic fungus Beauveria bassiana is used worldwide for biological control of insects. Seven dsRNA segments were detected in a single B. bassiana strain, RCEF1446. High-throughput sequencing indicated the presence of three mycoviruses in RCEF1446. Two were identified as the known mycoviruses Beauveria bassiana victorivirus 1 and Beauveria bassiana polymycovirus 1, and the novel mycovirus was designated as "Beauveria bassiana bipartite mycovirus 1" (BbBV1). The complete sequence of the BbBV1 is described here. The mycovirus contains two dsRNA segments. The RNA 1 (dsRNA 4) of BbBV1 is 2,026 bp in length, encoding a RNA-dependent RNA polymerase (RdRp) (68.54 kDa), while the RNA 2 (dsRNA 6) is 1,810 bp in length, encoding a hypothetical protein (35.55 kDa) with unknown function. Moreover, the amino acid sequence of RdRp showed the highest sequence identity of 62.31% to Botryosphaeria dothidea bipartite mycovirus 1. Phylogenetic analysis based on RdRp sequences revealed that BbBV1 represents a distinct lineage of unassigned dsRNA mycoviruses infecting fungi.


Assuntos
Beauveria/virologia , Vírus de RNA de Cadeia Dupla/genética , Micovírus/genética , Genoma Viral , Filogenia , Beauveria/patogenicidade , RNA de Cadeia Dupla , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética
13.
J Gen Virol ; 102(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34494948

RESUMO

Orbiviruses are arboviruses with 10 double-stranded linear RNA segments, and some have been identified as pathogens of dramatic epizootics in both wild and domestic ruminants. Tibet orbivirus (TIBOV) is a new orbivirus isolated from hematophagous insects in recent decades, and, currently, most of the strains have been isolated from insects in PR China, except for two from Japan. In this study, we isolated a novel reassortment TIBOV strain, YN15-283-01, from Culicoides spp. To identify and understand more characteristics of YN15-283-01, electrophoresis profiles of the viral genome, electron microscopic observations, plaque assays, growth curves in various cell lines, and bioinformatic analysis were conducted. The results indicated that YN15-283-01 replicated efficiently in mosquito cells, rodent cells and several primate cells. Furthermore, the maximum likelihood phylogenetic trees and simplot analysis of the 10 segments indicated that YN15-283-01 is a natural reassortment isolate that had emerged mainly from XZ0906 and SX-2017a.


Assuntos
Ceratopogonidae/virologia , Orbivirus/isolamento & purificação , Orbivirus/fisiologia , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/fisiologia , Animais , Linhagem Celular , China , Genoma Viral , Humanos , Orbivirus/classificação , Orbivirus/genética , Filogenia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Vírus Reordenados/classificação , Vírus Reordenados/genética , Replicação Viral
14.
J Biol Chem ; 297(4): 101218, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34562452

RESUMO

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.


Assuntos
Endorribonucleases/metabolismo , Modelos Moleculares , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Dimerização , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , SARS-CoV-2/isolamento & purificação , Transcrição Genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Replicação Viral
15.
Science ; 374(6567): eabj3624, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34581622
16.
Int J Mol Sci ; 22(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34575896

RESUMO

For the last 20 years, it has been common lore that the free energy of RNA duplexes formed from canonical Watson-Crick base pairs (bps) can be largely approximated with dinucleotide bp parameters and a few simple corrective constants that are duplex independent. Additionally, the standard benchmark set of duplexes used to generate the parameters were GC-rich in the shorter duplexes and AU-rich in the longer duplexes, and the length of the majority of the duplexes ranged between 6 and 8 bps. We were curious if other models would generate similar results and whether adding longer duplexes of 17 bps would affect the conclusions. We developed a gradient-descent fitting program for obtaining free-energy parameters-the changes in Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS), and the melting temperature (Tm)-directly from the experimental melting curves. Using gradient descent and a genetic algorithm, the duplex melting results were combined with the standard benchmark data to obtain bp parameters. Both the standard (Turner) model and a new model that includes length-dependent terms were tested. Both models could fit the standard benchmark data; however, the new model could handle longer sequences better. We developed an updated strategy for fitting the duplex melting data.


Assuntos
RNA de Cadeia Dupla/química , Algoritmos , Pareamento de Bases , Entropia , Modelos Lineares , Modelos Genéticos , Modelos Estatísticos , Modelos Teóricos , Distribuição Normal , Conformação de Ácido Nucleico , Temperatura , Termodinâmica
17.
Immunity ; 54(9): 1976-1988.e7, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34525338

RESUMO

Mutations in the adenosine-to-inosine RNA-editing enzyme ADAR1 p150, including point mutations in the Z-RNA recognition domain Zα, are associated with Aicardi-Goutières syndrome (AGS). Here, we examined the in vivo relevance of ADAR1 binding of Z-RNA. Mutation of W197 in Zα, which abolished Z-RNA binding, reduced RNA editing. Adar1W197A/W197A mice displayed severe growth retardation after birth, broad expression of interferon-stimulated genes (ISGs), and abnormal development of multiple organs. Notably, malformation of the brain was accompanied by white matter vacuolation and gliosis, reminiscent of AGS-associated encephalopathy. Concurrent deletion of the double-stranded RNA sensor MDA5 ameliorated these abnormalities. ADAR1 (W197A) expression increased in a feedback manner downstream of type I interferons, resulting in increased RNA editing at a subset of, but not all, ADAR1 target sites. This increased expression did not ameliorate inflammation in Adar1W197A/W197A mice. Thus, editing of select endogenous RNAs by ADAR1 is essential for preventing inappropriate MDA5-mediated inflammation, with relevance to the pathogenesis of AGS.


Assuntos
Adenosina Desaminase/genética , Doenças Autoimunes do Sistema Nervoso/genética , Malformações do Sistema Nervoso/genética , Edição de RNA/genética , RNA de Cadeia Dupla/genética , Adenosina Desaminase/metabolismo , Animais , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Modelos Animais de Doenças , Helicase IFIH1 Induzida por Interferon/metabolismo , Camundongos , Mutação , Malformações do Sistema Nervoso/fisiopatologia , RNA de Cadeia Dupla/metabolismo
18.
Immunity ; 54(9): 1961-1975.e5, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34525337

RESUMO

Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adenosina Desaminase/genética , Interferon Tipo I/imunologia , RNA de Cadeia Dupla/genética , Adenosina/genética , Adenosina/metabolismo , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Inosina/genética , Inosina/metabolismo , Interferon Tipo I/genética , Camundongos , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Edição de RNA/genética , RNA de Cadeia Dupla/metabolismo
19.
J Cell Sci ; 134(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34397095

RESUMO

Stress granules (SGs) are cytoplasmic assemblies of RNA and protein that form when translation is repressed during the integrated stress response. SGs assemble from the combination of RNA-RNA, RNA-protein and protein-protein interactions between messenger ribonucleoprotein complexes (mRNPs). The protein adenosine deaminase acting on RNA 1 (ADAR1, also known as ADAR) recognizes and modifies double-stranded RNAs (dsRNAs) within cells to prevent an aberrant innate immune response. ADAR1 localizes to SGs, and since RNA-RNA interactions contribute to SG assembly and dsRNA induces SGs, we examined how ADAR1 affects SG formation. First, we demonstrate that ADAR1 depletion triggers SGs by allowing endogenous dsRNA to activate the integrated stress response through activation of PKR (also known as EIF2AK2) and translation repression. However, we also show that ADAR1 limits SG formation independently of translation inhibition. ADAR1 repression of SGs is independent of deaminase activity but is dependent on dsRNA-binding activity, suggesting a model where ADAR1 binding limits RNA-RNA and/or RNA-protein interactions necessary for recruitment to SGs. Given that ADAR1 expression is induced during viral infection, these findings have implications for the role of ADAR1 in the antiviral response. This article has an associated First Person interview with the first author of the paper.


Assuntos
Adenosina Desaminase , Imunidade Inata , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Grânulos Citoplasmáticos/metabolismo , Humanos , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética
20.
J Virol ; 95(20): e0113421, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34346771

RESUMO

Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.


Assuntos
Infecções por Caliciviridae/imunologia , Macrófagos/virologia , Fator 2 Ativador da Transcrição/metabolismo , Animais , Antivirais , Infecções por Caliciviridae/metabolismo , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Imunidade Inata/imunologia , Inflamação/imunologia , Interferons , Macrófagos/imunologia , Camundongos , Norovirus/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7 , RNA de Cadeia Dupla/genética , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genética
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