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1.
J Virol ; 98(5): e0017724, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38563731

RESUMO

Cactin, a highly conserved protein, plays a crucial role in various physiological processes in eukaryotes, including innate immunity. Recently, the function of Cactin in the innate immunity of Drosophila has been explored, revealing that Cactin regulates a non-canonical signaling pathway associated with the Toll and Imd pathways via the Cactin-Deaf1 axis. In addition, Cactin exhibits specific antiviral activity against the Drosophila C virus (DCV) in Drosophila, with an unknown mechanism. During DCV infection, it has been confirmed that the protein level and antiviral activity of Cactin are regulated by ubiquitination. However, the precise ubiquitination and deubiquitination mechanisms of Cactin in Drosophila remain unexplored. In this study, we identified ubiquitin-specific protease 14 (Usp14) as a major deubiquitinase for Cactin through comprehensive deubiquitinase screening. Our results demonstrate that Usp14 interacts with the C_Cactus domain of Cactin via its USP domain. Usp14 efficiently removes K48- and K63-linked polyubiquitin chains from Cactin, thereby preventing its degradation through the ubiquitin-proteasome pathway. Usp14 significantly inhibits DCV replication in Drosophila cells by stabilizing Cactin. Moreover, Usp14-deficient fruit flies exhibit increased susceptibility to DCV infection compared to wild-type flies. Collectively, our findings reveal the regulation of ubiquitination and antiviral activity of Cactin by the deubiquitinase Usp14, providing valuable insights into the modulation of Cactin-mediated antiviral activity in Drosophila.IMPORTANCEViral infections pose a severe threat to human health, marked by high pathogenicity and mortality rates. Innate antiviral pathways, such as Toll, Imd, and JAK-STAT, are generally conserved across insects and mammals. Recently, the multi-functionality of Cactin in innate immunity has been identified in Drosophila. In addition to regulating a non-canonical signaling pathway through the Cactin-Deaf1 axis, Cactin exhibits specialized antiviral activity against the Drosophila C virus (DCV) with an unknown mechanism. A previous study emphasized the significance of the Cactin level, regulated by the ubiquitin-proteasome pathway, in modulating antiviral signaling. However, the regulatory mechanisms governing Cactin remain unexplored. In this study, we demonstrate that Usp14 stabilizes Cactin by preventing its ubiquitination and subsequent degradation. Furthermore, Usp14 plays a crucial role in regulating the antiviral function mediated by Cactin. Therefore, our findings elucidate the regulatory mechanism of Cactin in Drosophila, offering a potential target for the prevention and treatment of viral infections.


Assuntos
Proteínas de Drosophila , Imunidade Inata , Ubiquitinação , Animais , Dicistroviridae/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/virologia , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Replicação Viral
2.
PLoS Pathog ; 19(3): e1011238, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36961862

RESUMO

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.


Assuntos
Afídeos , Oryza , Vírus , Animais , Humanos , Oryza/genética , Afídeos/genética , Perfilação da Expressão Gênica , Plantas Geneticamente Modificadas/genética , Vírus/genética , Doenças das Plantas
3.
Plant Cell Environ ; 2024 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-39390751

RESUMO

Pandemics originating from zoonotic viruses have posed significant threats to human health and agriculture. Recent discoveries have revealed that wild-rice plants also harbour viral pathogens capable of severely impacting rice production, a cornerstone food crop. In this study, we conducted virome analysis on ~1000 wild-rice individual colonies and discovered a novel single-strand positive-sense RNA virus prevalent in these plants. Through comprehensive genomic characterization and comparative sequence analysis, this virus was classified as a new species in the genus Polerovirus, designated Rice less tiller virus (RLTV). Our investigations elucidated that RLTV could be transmitted from wild rice to cultivated rice via a specific insect vector, the aphid Rhopalosiphum padi, causing less tiller disease symptoms in rice plants. We generated an infectious cDNA clone for RLTV and demonstrated systemic infection of rice cultivars and induction of severe disease symptoms following mechanical inoculation or stable genetic transformation. We further illustrated transmission of RLTV from stable transgenic lines to healthy rice plants by the aphid vector, leading to the development of disease symptoms. Notably, our database searches showed that RLTV and another polerovirus isolated from a wild plant species are widely circulating not only in wild rice but also cultivated rice around the world. Our findings provide strong evidence for a wild plant origin for rice viruses and underscore the imminent threat posed by aphid-transmitted rice Polerovirus to rice cultivar.

4.
Genomics ; 115(6): 110729, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37852332

RESUMO

Sogatella furcifera is a destructive agricultural pest causing large threats to rice production in China and Southeast Asian countries. Despite recent breakthroughs in long-read sequencing, high quality genomic data are very limited in S. furcifera. In present study, a chromosome-level assembly of the S. furcifera genome was completed (0.64 GB), comprising 15 chromosomes covered 95.04% of the estimated genome size, along with other 624 small scaffolds making up the remaining 4.96% of the genome of S. furcifera. A total of 24,669 protein-coding genes, 1211 long noncoding RNA and 7595 circular RNA transcripts were predicted in this study. Comparative genomic analysis revealed rapidly evolved genes were associated with multiple immune-related pathways in S. furcifera. Genome resequencing of 44 individuals from 12 geographic populations revealed frequent gene flow among populations. The systemic genomic analysis will provide more insights into the understanding of the immunity and evolutionary adaptation of S. furcifera.


Assuntos
Hemípteros , Metagenômica , Humanos , Animais , Genômica , China , Ásia , Hemípteros/genética , Cromossomos
5.
Arch Virol ; 167(12): 2811-2815, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36269416

RESUMO

The complete genome of a novel virus, provisionally named "Camellia sinensis badnavirus 1" (CSBV1), was identified in tea plant (Camellia sinensis) leaves collected in Anhui Province, China. The genome of CSBV1 consists of 8,195 bp and possesses three open reading frames (ORFs), sharing 68.6 % nucleotide sequence identity with the genome of Camellia lemon glow virus (CLGV) from Camellia japonica. The genome organization of CSBV1 is highly similar to that of members of the genus Badnavirus (family Caulimoviridae). Phylogenetic analysis revealed that CSBV1, CLGV, and cacao swollen shoot virus form a separate clade within the genus Badnavirus, suggesting that CSBV1 is the first badnavirus infecting C. sinensis.


Assuntos
Badnavirus , Camellia sinensis , Filogenia , Camellia sinensis/genética , Genoma Viral , Doenças das Plantas , Badnavirus/genética , Fases de Leitura Aberta
6.
BMC Genomics ; 20(1): 396, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113376

RESUMO

BACKGROUND: Phenotypic plasticity is a common and highly adaptive phenomenon where the same genotype produces different phenotypes in response to environmental cues. Sogatella furcifera, a migratory pest of rice exhibits wing dimorphism, is a model insect for studying phenotypic plasticity of wing size. The Insullin-PI3K-Akt-FOXO signaling pathway plays a crucial role in the manipulation of wing size in the migratory insects. However, the regulatory mechanism via the pathway involved in wing dimorphism are still unexplored. RESULTS: Accompanied by special alternative splicing, genes involved in muscle contraction and energy metabolism were highly expressed in the wing hinges of macropters, demonstrating their adaptation for energy-demanding long-distance flights. Based on FOXO ChIP-Seq analysis, a total of 1259 putative target genes were observed in the wing hinges, including wing morph development, flight muscle and energy metabolism genes. An integrated gene interaction network was built by combining four heterogeneous datasets, and the IIS-PI3K-Akt-FOXO pathway was clustered in a divided functional module. In total, 45 genes in the module directly interacting with the IIS-PI3K-Akt-FOXO pathway showed differential expression levels between the two wing hinges, thus are regarded as potential Insulin pathway mediated wing dimorphism related genes (IWDRGs). Of the 45 IWDRGs, 5 were selected for verification by gene knockdown experiments, and played significant roles in the insect wing size regulation. CONCLUSIONS: We provided valuable insights on the genetic basis of wing dimorphism, and also demonstrated that network analysis is a powerful approach to identify new genes regulating wing dimorphic development via insulin signaling pathway in the migratory insect.


Assuntos
Genes de Insetos , Hemípteros/genética , Insulina/fisiologia , Asas de Animais/metabolismo , Processamento Alternativo , Animais , Ácidos Graxos/metabolismo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Hemípteros/anatomia & histologia , Hemípteros/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Musculares/genética , Fenótipo , Transdução de Sinais , Asas de Animais/anatomia & histologia
7.
BMC Genomics ; 19(1): 688, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30231855

RESUMO

BACKGROUND: Sogatella furcifera is a migratory pest that damages rice plants and causes severe economic losses. Due to its ability to annually migrate long distances, S. furcifera has emerged as a major pest of rice in several Asian countries. Symbiotic relationships of inherited bacteria with terrestrial arthropods have significant implications. The genus Cardinium is present in many types of arthropods, where it influences some host characteristics. We present a report of a newly identified strain of the bacterial endosymbiont Cardinium cSfur in S. furcifera. RESULT: From the whole genome of S. furcifera previously sequenced by our laboratory, we assembled the whole genome sequence of Cardinium cSfur. The sequence comprised 1,103,593 bp with a GC content of 39.2%. The phylogenetic tree of the Bacteroides phylum to which Cardinium cSfur belongs suggests that Cardinium cSfur is closely related to the other strains (Cardinium cBtQ1 and cEper1) that are members of the Amoebophilaceae family. Genome comparison between the host-dependent endosymbiont including Cardinium cSfur and free-living bacteria revealed that the endosymbiont has a smaller genome size and lower GC content, and has lost some genes related to metabolism because of its special environment, which is similar to the genome pattern observed in other insect symbionts. Cardinium cSfur has limited metabolic capability, which makes it less contributive to metabolic and biosynthetic processes in its host. From our findings, we inferred that, to compensate for its limited metabolic capability, Cardinium cSfur harbors a relatively high proportion of transport proteins, which might act as the hub between it and its host. With its acquisition of the whole operon related to biotin synthesis and glycolysis related genes through HGT event, Cardinium cSfur seems to be undergoing changes while establishing a symbiotic relationship with its host. CONCLUSION: A novel bacterial endosymbiont strain (Cardinium cSfur) has been discovered. A genomic analysis of the endosymbiont in S. furcifera suggests that its genome has undergone certain changes to facilitate its settlement in the host. The envisaged potential reproduction manipulative ability of the new endosymbiont strain in its S. furcifera host has vital implications in designing eco-friendly approaches to combat the insect pest.


Assuntos
Proteínas de Bactérias/genética , Cytophagaceae/fisiologia , Genoma Bacteriano , Hemípteros/genética , Hemípteros/microbiologia , Simbiose/fisiologia , Animais , Genômica , Hemípteros/crescimento & desenvolvimento , Filogenia
8.
Arch Virol ; 163(10): 2929-2931, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30022239

RESUMO

Maize-associated totivirus Anhui (MATV-Ah) is a novel totivirus with a 5536-nt genome and two large ORFs that encode a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRP). The two ORFs share amino acid identities of 32 and 56% when compared to other plant-associated totiviruses, respectively. Based on genome sequence similarity and phylogenetic analysis, MATV-Ah is proposed to be a member of the family Totiviridae genus Totivirus.


Assuntos
Genoma Viral , Doenças das Plantas/virologia , Totivirus/isolamento & purificação , Zea mays/virologia , China , Fases de Leitura Aberta , Filogenia , Totivirus/classificação , Totivirus/genética
9.
Nucleic Acids Res ; 44(8): 3936-45, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-26939889

RESUMO

Despite over 3300 protein-DNA complex structures have been reported in the past decades, there remain some unknown recognition patterns between protein and target DNA. The silkgland-specific transcription factor FMBP-1 from the silkworm Bombyx mori contains a unique DNA-binding domain of four tandem STPRs, namely the score and three amino acid peptide repeats. Here we report three structures of this STPR domain (termed BmSTPR) in complex with DNA of various lengths. In the presence of target DNA, BmSTPR adopts a zig-zag structure of three or four tandem α-helices that run along the major groove of DNA. Structural analyses combined with binding assays indicate BmSTPR prefers the AT-rich sequences, with each α-helix covering a DNA sequence of 4 bp. The successive AT-rich DNAs adopt a wider major groove, which is in complementary in shape and size to the tandem α-helices of BmSTPR. Substitutions of DNA sequences and affinity comparison further prove that BmSTPR recognizes the major groove mainly via shape readout. Multiple-sequence alignment suggests this unique DNA-binding pattern should be highly conserved for the STPR domain containing proteins which are widespread in animals. Together, our findings provide structural insights into the specific interactions between a novel DNA-binding protein and a unique deformed B-DNA.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Insetos/química , Fatores de Transcrição/química , Animais , Sítios de Ligação , Bombyx , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Insetos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/metabolismo
10.
PLoS Pathog ; 10(12): e1004553, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25503469

RESUMO

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.


Assuntos
Algoritmos , Biologia Computacional/métodos , RNA Viral/genética , RNA/genética , Análise de Sequência de RNA/métodos , Viroides/genética , Sequência de Bases , Malus , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Circular , Viroides/fisiologia , Replicação Viral/fisiologia , Vitis
11.
Int J Med Microbiol ; 306(8): 686-696, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27600408

RESUMO

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR associated proteins [Cas]) system can provide prokaryote with immunity against invading mobile genetic elements (MGEs) such as phages and plasmids, which are the main sources of staphylococcal accessory genes. To date, only a few Staphylococcus aureus strains containing CRISPR-Cas systems have been identified, but no functional study in these strains has been reported. In this study, 6 clinical isolates of S. aureus with type III-A CRISPR-Cas systems were identified, and whole-genome sequencing and functional study were conducted subsequently. Genome sequence analysis revealed a close linkage between the CRISPR-Cas system and the staphylococcal cassette chromosome mec (SCCmec) element in five strains. Comparative sequence analysis showed that the type III-A repeats are conserved within staphylococci, despite of the decreased conservation in trailer-end repeats. Highly homologous sequences of some spacers were identified in staphylococcal MGEs, and partially complementary sequences of spacers were mostly found in the coding strand of lytic regions in staphylococcal phages. Transformation experiments showed that S. aureus type III-A CRISPR-Cas system can specifically prevent plasmid transfer in a transcription-dependent manner. Base paring between crRNA and target sequence, the endoribonuclease, and the Csm complex were proved to be necessary for type III-A CRISPR-Cas immunity.


Assuntos
Sistemas CRISPR-Cas , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Ordem dos Genes , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Transcrição Gênica , Transformação Bacteriana
12.
Virol J ; 13(1): 181, 2016 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-27814723

RESUMO

BACKGROUND: The invasion of plant by viruses cause major damage to plants and reduces crop yield and integrity. Devastating plant virus infection has been experienced at different times all over the world, which are attributed to different events of mutation, re-assortment and recombination occurring in the viruses. Strategies for proper virus management has been mostly limited to eradicating the vectors that spreads the plant viruses. However, development of prompt and effective diagnostic methods are required to monitor emerging and re-emerging diseases that may be symptomatic or asymptomatic in the plant as well as the genetic variation and evolution in the plant viruses. A survey of plant viruses infecting field-grown Tobacco crop was conducted in Anhui Province of China by the deep sequencing of sRNAs. METHODS: Survey of plant viruses infecting Tobacco was carried based on 104 samples collected across the province. Nine different sRNA libraries was prepared and custom-made bioinformatics pipeline coupled with molecular techniques was developed to sequence, assemble and analyze the siRNAs for plant virus discovery. We also carried out phylogenetic and recombination analysis of the identified viruses. RESULTS: Twenty two isolates from eight different virus species including Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, Tobacco vein banding Mosaic virus, Pepper mottle virus, Brassica yellow virus, Chilli venial mottle virus, Broad bean wilt virus 2 were identified in tobacco across the survey area. The near-complete genome sequence of the 22 new isolates were determined and analyzed. The isolates were grouped together with known strains in the phylogenetic tree. Molecular variation in the isolates indicated the conserved coding regions have majorly a nucleotide sequence identity of 80-94 % with previously identified isolates. Various events of recombination were discovered among some of the isolates indicating that two or more viruses or different isolates of one virus infect the same host cell. CONCLUSION: This study describes the discovery of a consortium of plant viruses infecting Tobacco that are broadly distributed in Anhui province of China. It also demonstrates the effectiveness of NGS in identifying plant viruses without a prior knowledge of the virus and the genetic diversity that enhanced mixed infection.


Assuntos
Ecótipo , Variação Genética , Nicotiana/virologia , Vírus de Plantas/classificação , Vírus de Plantas/isolamento & purificação , China , Biologia Computacional , Metagenômica , Vírus de Plantas/genética , Análise de Sequência de DNA
13.
J Gen Virol ; 96(8): 2411-2420, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25934791

RESUMO

A novel circular DNA virus sequence has been identified through next-generation sequencing and in silico assembly of small RNAs of 21-24 nt from an apple tree grown in China. The virus genome was cloned using two independent approaches and sequenced. With a size of 2932 nt, it showed the same genomic structure and conserved origin of replication reported for members of the family Geminiviridae. However, the low nucleotide and amino acid sequence identity with known geminiviruses indicated that it was a novel virus, for which the provisional name apple geminivirus (AGV) is proposed. Rolling circle amplification followed by RFLP analyses indicated that AGV was a virus with a monopartite DNA genome. This result was in line with bioassays showing that the cloned viral genome was infectious in several herbaceous plants (Nicotiana bethamiana, Nicotiana glutinosa and Solanum lycopersicum), thus confirming it was complete and biologically active, although no symptoms were observed in these experimental hosts. AGV genome structure and phylogenetic analyses did not support the inclusion of this novel species in any of the established genera in the family Geminiviridae. A survey of 165 apple trees grown in four Chinese provinces showed a prevalence of 7.2% for AGV, confirming its presence in several cultivars and geographical areas in China, although no obvious relationship between virus infection and specific symptoms was found.


Assuntos
Geminiviridae/genética , Geminiviridae/isolamento & purificação , Genoma Viral , Malus/virologia , Doenças das Plantas/virologia , Sequência de Aminoácidos , China , Geminiviridae/química , Geminiviridae/classificação , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Biochem Biophys Res Commun ; 460(2): 222-6, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25770426

RESUMO

Disconnected Interacting Protein 1 (DIP1) is a dsRNA-binding protein that participates in a wide range of cellular processes. Whether DIP1 is involved in innate immunity remains unclear. Here, DIP1 was found to play an antiviral role in S2 cells. Its antiviral action is specific for DCV infection and not for DXV infection. dip1 mutant flies are hypersensitive to DCV infection. The increased mortality in dip1 mutant flies is associated with the accumulation of DCV positive-stranded RNAs in vivo. This study demonstrated that dip1 is a novel antiviral gene that restricts DCV replication in vitro and in vivo.


Assuntos
Proteínas de Drosophila/fisiologia , Vírus de Insetos/patogenicidade , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Drosophila melanogaster , Viroses/fisiopatologia , Viroses/prevenção & controle
15.
Arch Virol ; 160(9): 2367-70, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26088445

RESUMO

The complete genome of a novel virus, provisionally named areca palm velarivirus 1 (APV1), was identified in areca palm exhibiting leaf yellowing symptoms in Hainan province, China. The genome of APV1 consists of 16,080 nucleotides and possesses 11 open reading frames (ORFs), sharing 56.4% nucleotide sequence identity with little cherry virus 1 (NC_001836.1). The genome organization of APV1 is highly similar to that of members of the genus Velarivirus (family Closteroviridae). Phylogenetic analysis placed APV1 together with members of the genus Velarivirus.


Assuntos
Closteroviridae/genética , Genoma Viral , RNA Viral/genética , Análise de Sequência de DNA , Areca/virologia , China , Closteroviridae/isolamento & purificação , Análise por Conglomerados , Ordem dos Genes , Fases de Leitura Aberta , Filogenia , Doenças das Plantas/virologia , Homologia de Sequência do Ácido Nucleico
16.
Proc Natl Acad Sci U S A ; 109(10): 3938-43, 2012 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-22345560

RESUMO

A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their full-length sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed.


Assuntos
Biologia Computacional/métodos , RNA/genética , Algoritmos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Modelos Genéticos , Conformação de Ácido Nucleico , Doenças das Plantas/virologia , RNA Catalítico/química , RNA Catalítico/genética , RNA Circular , RNA Viral/genética , Software , Viroides/química , Replicação Viral , Vitis/virologia
17.
Plant Cell ; 23(4): 1625-38, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21467580

RESUMO

Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/virologia , Cucumovirus/imunologia , Nucleotídeos/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Antivirais/imunologia , Arabidopsis/genética , Proteínas Argonautas , Inativação Gênica , Imunidade Inata/genética , Imunidade Inata/imunologia , Mutação/genética , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Ligação Proteica
18.
mBio ; 15(9): e0146924, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39158293

RESUMO

RNA interference (RNAi) drives powerful antiviral immunity in plants and animals so that many viruses must express viral suppressor of RNAi (VSR) to establish virulent infection. However, little is known about the immune responses conferring resistance against viruses that have evolved the counter-defensive strategy to suppress antiviral RNAi. In this study, we discover that Drosophila cells infected with Drosophila C virus (DCV), a natural viral pathogen of Drosophila known to harbor a potent VSR, exhibit heightened expression of circular RNA circZfh1. circZfh1 confers virus resistance in the presence of viral suppression of antiviral RNAi. Furthermore, we validate that circZfh1 encodes a 274-amino acid protein, CRAV, essential for its antiviral activity. Notably, CRAV differs from its parental Zfh1 gene in a different reading frame, with the C-terminal 69 amino acids unique to CRAV. Our analysis also reveals the presence of CRAV in species within the melanogaster subgroup, with the C-terminal unique fragment undergoing accelerated evolution. Expression of CRAV upregulates the expression of the cytokine Upd3, which binds to its receptor, stimulating the JAK-STAT pathway and enhancing the immune response to DCV infection. Notably, CRISPR/Cas9 knockout of circZfh1 significantly enhances DCV replication in vitro and in vivo, with circZfh1-knockout adult flies displaying heightened disease susceptibility to DCV. In summary, our findings unveil a Drosophila protein-coding circular RNA that activates an innate immune signaling pathway crucial for virus resistance following the suppression of antiviral RNAi by viruses, thereby elucidating a novel counter-defensive strategy.IMPORTANCEEukaryotic hosts possess a complex, multilayered immune system that guards against pathogen invasion. In fruit flies, RNA interference (RNAi) drives robust antiviral immunity, prompting many viruses to express viral suppressors of RNAi (VSRs) to establish virulent infections. However, little is known about immune responses that confer resistance against viruses with potent VSRs. In this study, we discovered that Drosophila cells infected with Drosophila C virus (DCV), a natural viral pathogen possessing a potent VSR, upregulated the expression of circular RNA circZfh1. circZfh1 exhibits DCV-specific antiviral activity, encoding a 274-amino acid protein, CRAV, crucial for its antiviral effects. As a different reading frame from its parental Zfh1 gene, the C-terminal 69 amino acids are unique to CRAV, undergoing faster evolution. CRAV activates the JAK-STAT pathway, enhancing the immune response to DCV infection. Therefore, our work uncovers a new strategy for suppressing viral counter-defense through protein-coding circular RNA in fruit flies.


Assuntos
Dicistroviridae , Proteínas de Drosophila , Drosophila melanogaster , Janus Quinases , RNA Circular , Fatores de Transcrição STAT , Animais , RNA Circular/genética , RNA Circular/imunologia , Janus Quinases/metabolismo , Janus Quinases/genética , Janus Quinases/imunologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/genética , Drosophila melanogaster/virologia , Dicistroviridae/genética , Dicistroviridae/imunologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição STAT/imunologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/imunologia , Imunidade Inata/genética , Transdução de Sinais , Interferência de RNA , Drosophila/genética , Drosophila/imunologia , Drosophila/virologia , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/genética
19.
Front Mol Biosci ; 11: 1399679, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38831933

RESUMO

Background: Gastric cancer is a highly prevalent malignant neoplasm. Metabolic reprogramming is intricately linked to both tumorigenesis and cancer immune evasion. The advent of single-cell RNA sequencing technology provides a novel perspective for evaluating cellular metabolism. This study aims to comprehensively investigate the metabolic pathways of various cell types in tumor and normal samples at high resolution and delve into the intricate regulatory mechanisms governing the metabolic activity of malignant cells in gastric cancer. Methods: Utilizing single-cell RNA sequencing data from gastric cancer, we constructed metabolic landscape maps for different cell types in tumor and normal samples. Employing unsupervised clustering, we categorized malignant cells in tumor samples into high and low metabolic subclusters and further explored the characteristics of these subclusters. Results: Our research findings indicate that epithelial cells in tumor samples exhibit significantly higher activity in most KEGG metabolic pathways compared to other cell types. Unsupervised clustering, based on the scores of metabolic pathways, classified malignant cells into high and low metabolic subclusters. In the high metabolic subcluster, it demonstrated the potential to induce a stronger immune response, correlating with a relatively favorable prognosis. In the low metabolic subcluster, a subset of cells resembling cancer stem cells (CSCs) was identified, and its prognosis was less favorable. Furthermore, a set of risk genes associated with this subcluster was discovered. Conclusion: This study reveals the intricate regulatory mechanisms governing the metabolic activity of malignant cells in gastric cancer, offering new perspectives for improving prognosis and treatment strategies.

20.
mBio ; 15(4): e0137823, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38446061

RESUMO

Liquid-liquid phase separation (LLPS) plays a crucial role in various biological processes in eukaryotic organisms, including immune responses in mammals. However, the specific function of LLPS in immune responses in Drosophila melanogaster remains poorly understood. Cactin, a highly conserved protein in eukaryotes, is involved in a non-canonical signaling pathway associated with Nuclear factor-κB (NF-κB)-related pathways in Drosophila. In this study, we investigated the role of Cactin in LLPS and its implications for immune response modulation. We discovered that Cactin undergoes LLPS, forming droplet-like particles, primarily mediated by its intrinsically disordered region (IDR). Utilizing immunoprecipitation and mass spectrometry analysis, we identified two phosphorylation sites at serine residues 99 and 104 within the IDR1 domain of Cactin. Co-immunoprecipitation and mass spectrometry further revealed phosphoglycerate kinase (PGK) as a Cactin-interacting protein responsible for regulating its phosphorylation. Phosphorylation of Cactin by PGK induced a transition from stable aggregates to dynamic liquid droplets, enhancing its ability to interact with other components in the cellular environment. Overexpression of PGK inhibited Drosophila C virus (DCV) replication, while PGK knockdown increased replication. DCV infection also increased Cactin phosphorylation. We also found that phosphorylation enhances the antiviral ability of Cactin by promoting liquid-phase droplet formation. These findings demonstrate the role of Cactin-phase separation in regulating DCV replication and highlight the modulation of its antiviral function through phosphorylation, providing insights into the interplay between LLPS and antiviral defense mechanisms. IMPORTANCE: Liquid-liquid phase separation (LLPS) plays an integral role in various biological processes in eukaryotic organisms. Although several studies have highlighted its crucial role in modulating immune responses in mammals, its function in immune responses in Drosophila melanogaster remains poorly understood. Our study investigated the role of Cactin in LLPS and its implications for immune response modulation. We identified that phosphoglycerate kinase (PGK), an essential enzyme in the glycolytic pathway, phosphorylates Cactin, facilitating its transition from a relatively stable aggregated state to a more dynamic liquid droplet phase during the phase separation process. This transformation allows Cactin to rapidly interact with other cellular components, enhancing its antiviral properties and ultimately inhibiting virus replication. These findings expand our understanding of the role of LLPS in the antiviral defense mechanism, shedding light on the intricate mechanisms underlying immune responses in D. melanogaster.


Assuntos
Proteínas de Transporte , Proteínas de Drosophila , Drosophila melanogaster , Drosophila , Fosfoglicerato Quinase , Animais , Separação de Fases , Fosforilação
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