RESUMEN
Arboviruses can be paternally transmitted by male insects to offspring for long-term persistence, but the mechanism remains largely unknown. Here, we use a model system of a destructive rice reovirus and its leafhopper vector to find that insect ribosome-rescuer Pelo-Hbs1 complex expressed on the sperm surface mediates paternal arbovirus transmission. This occurs through targeting virus-containing tubules constituted by viral nonstructural protein Pns11 to sperm surface via Pns11-Pelo interaction. Tubule assembly is dependent on Hsp70 activity, while Pelo-Hbs1 complex inhibits tubule assembly via suppressing Hsp70 activity. However, virus-activated ubiquitin ligase E3 mediates Pelo ubiquitinated degradation, synergistically causing Hbs1 degradation. Importantly, Pns11 effectively competes with Pelo for binding to E3, thus antagonizing E3-mediated Pelo-Hbs1 degradation. These processes cause a slight reduction of Pelo-Hbs1 complex in infected testes, promoting effective tubule assembly. Our findings provide insight into how insect sperm-specific Pelo-Hbs1 complex is modulated to promote paternal virus transmission without disrupting sperm function.
Asunto(s)
Hemípteros , Proteínas de Insectos , Espermatozoides , Animales , Masculino , Espermatozoides/metabolismo , Espermatozoides/virología , Hemípteros/virología , Hemípteros/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Arbovirus , Proteínas HSP70 de Choque Térmico/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Reoviridae/fisiología , Insectos Vectores/virología , Insectos Vectores/metabolismo , Ribosomas/metabolismo , Infecciones por Arbovirus/transmisión , Infecciones por Arbovirus/metabolismo , Infecciones por Arbovirus/virologíaRESUMEN
Reovirus (Reo) has shown promising potential in specifically killing tumor cells, and offering new possibilities for ovarian cancer (OC) treatment. However, neutralizing antibodies in the ascites from OC patients greatly limit the further application of Reo. In this study, we employed cationic liposomes (Lipo) to deliver Reo, significantly enhancing its ability to enter OC cells and its effectiveness in killing these cells under ascitic conditions. Pre-treatment with the MßCD inhibitor notably decreased Reo-mediated tumor cell death, indicating that Lipo primarily enables Reo's cellular uptake through caveolin-mediated endocytosis. Our results demonstrate that Lipo effectively facilitates the entry of Reo into the cytoplasm and triggers cell apoptosis. The above findings provide a new strategy to overcome the obstacle of neutralizing antibodies in the clinical application of Reo.
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Anticuerpos Neutralizantes , Liposomas , Neoplasias Ováricas , Reoviridae , Femenino , Humanos , Neoplasias Ováricas/inmunología , Anticuerpos Neutralizantes/inmunología , Reoviridae/inmunología , Reoviridae/fisiología , Línea Celular Tumoral , Viroterapia Oncolítica/métodos , Apoptosis , Animales , Cationes , Virus Oncolíticos/inmunología , RatonesRESUMEN
Salivary proteins of insect herbivores can suppress plant defenses, but the roles of many remain elusive. One such protein is glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the saliva of the Recilia dorsalis (RdGAPDH) leafhopper, which is known to transmit rice gall dwarf virus (RGDV). Here we show that RdGAPDH was loaded into exosomes and released from salivary glands into the rice phloem through an exosomal pathway as R. dorsalis fed. In infected salivary glands of R. dorsalis, the virus upregulated the accumulation and subsequent release of exosomal RdGAPDH into the phloem. Once released, RdGAPDH consumed H2O2 in rice plants owing to its -SH groups reacting with H2O2. This reduction in H2O2 of rice plant facilitated R. dorsalis feeding and consequently promoted RGDV transmission. However, overoxidation of RdGAPDH could cause potential irreversible cytotoxicity to rice plants. In response, rice launched emergency defense by utilizing glutathione to S-glutathionylate the oxidization products of RdGAPDH. This process counteracts the potential cellular damage from RdGAPDH overoxidation, helping plant to maintain a normal phenotype. Additionally, salivary GAPDHs from other hemipterans vectors similarly suppressed H2O2 burst in plants. We propose a strategy by which plant viruses exploit insect salivary proteins to modulate plant defenses, thus enabling sustainable insect feeding and facilitating viral transmission.
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Hemípteros , Peróxido de Hidrógeno , Oryza , Enfermedades de las Plantas , Saliva , Animales , Hemípteros/virología , Peróxido de Hidrógeno/metabolismo , Oryza/virología , Oryza/metabolismo , Enfermedades de las Plantas/virología , Saliva/metabolismo , Saliva/virología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glándulas Salivales/virología , Glándulas Salivales/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Insectos Vectores/virología , Floema/virología , Floema/metabolismo , Reoviridae/fisiología , Glutatión/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Virus de Plantas/fisiología , Defensa de la Planta contra la HerbivoriaRESUMEN
Rice black-streaked dwarf virus (RBSDV) is an etiological agent of a destructive disease infecting some economically important crops from the Gramineae family in Asia. While RBSDV causes high yield losses, genetic characteristics of replicative viral populations have not been investigated within different host plants and insect vectors. Herein, eleven publicly available RNA-Seq datasets from Chinese RBSDV-infected rice, maize, and viruliferous planthopper (Laodelphax striatellus) were obtained from the NCBI database. The patterns of SNP and RNA expression profiles of expected RBSDV populations were analyzed by CLC Workbench 20 and Geneious Prime software. These analyses discovered 2,646 mutations with codon changes in RBSDV whole transcriptome and forty-seven co-mutated hotspots with high variant frequency within the crucial regions of S5-1, S5-2, S6, S7-1, S7-2, S9, and S10 open reading frames (ORFs) which are responsible for some virulence and host range functions. Moreover, three joint mutations are located on the three-dimensional protein of P9-1. The infected RBSDV-susceptible rice cultivar KTWYJ3 and indigenous planthopper datasets showed more co-mutated hotspot numbers than others. Our analyses showed the expression patterns of viral genomic fragments varied depending on the host type. Unlike planthopper, S5-1, S2, S6, and S9-1 ORFs, respectively had the greatest read numbers in host plants; and S5-2, S9-2, and S7-2 were expressed in the lowest level. These findings underscore virus/host complexes are effective in the genetic variations and gene expression profiles of plant viruses. Our analysis revealed no evidence of recombination events. Interestingly, the negative selection was observed at 12 RBSDV ORFs, except for position 1015 in the P1 protein, where a positive selection was detected. The research highlights the potential of SRA datasets for analysis of the virus cycle and enhances our understanding of RBSDV's genetic diversity and host specificity.
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Insectos Vectores , Oryza , Enfermedades de las Plantas , Virus de Plantas , Animales , Oryza/virología , Oryza/genética , Insectos Vectores/virología , Insectos Vectores/genética , Virus de Plantas/genética , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Hemípteros/virología , Hemípteros/genética , Variación Genética , RNA-Seq , Transcriptoma , Reoviridae/genética , Zea mays/virología , Zea mays/genética , Polimorfismo de Nucleótido Simple , Mutación , Perfilación de la Expresión Génica , Sistemas de Lectura Abierta/genéticaRESUMEN
Similar to other RNA viruses, grass carp reovirus, the causative agent of the hemorrhagic disease, replicates in cytoplasmic viral inclusion bodies (VIBs), orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of GCRV VIBs are poorly understood. This work demonstrates that GCRV manipulates grass carp oxysterol binding protein 1 (named as gcOSBP1) and vesicle-associated membrane protein-associated protein A/B (named as gcVAP-A/B), 3 components of cholesterol transport pathway, to generate VIBs. By siRNA-mediated knockdown, we demonstrate that gcOSBP1 is an essential host factor for GCRV replication. We reveal that the nonstructural proteins NS80 and NS38 of GCRV interact with gcOSBP1, and that the gcOSBP1 is recruited by NS38 and NS80 for promoting the generation of VIBs. gcOSBP1 increases the expression of gcVAP-A/B and promotes the accumulation of intracellular cholesterol. gcOSBP1 also interacts with gcVAP-A/B for forming gcOSBP1-gcVAP-A/B complexes, which contribute to enhance the accumulation of intracellular cholesterol and gcOSBP1-mediated generation of VIBs. Inhibiting cholesterol accumulation by lovastatin can completely abolish the effects of gcOSBP1 and/or gcVAP-A/B in promoting GCRV infection, suggesting that cholesterol accumulation is vital for gcOSBP1- and/or gcVAP-A/B-mediated GCRV replication. Thus, our results, which highlight that gcOSBP1 functions in the replication of GCRV via its interaction with essential viral proteins for forming VIBs and with host gcVAP-A/B, provide key molecular targets for obtaining anti-hemorrhagic disease grass carp via gene editing technology.
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Carpas , Colesterol , Cuerpos de Inclusión Viral , Receptores de Esteroides , Reoviridae , Replicación Viral , Animales , Reoviridae/fisiología , Carpas/virología , Carpas/metabolismo , Cuerpos de Inclusión Viral/metabolismo , Colesterol/metabolismo , Receptores de Esteroides/metabolismo , Enfermedades de los Peces/virología , Enfermedades de los Peces/metabolismo , Enfermedades de los Peces/inmunología , Interacciones Huésped-Patógeno , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/metabolismo , Infecciones por Reoviridae/virología , Proteínas de Peces/metabolismo , Proteínas de Peces/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genéticaRESUMEN
In addition to chemotherapy, oncolytic viruses are an efficient treatment for acute myeloid leukemia (AML). Like other oncolytic viruses, the anti-tumor efficacy of reovirus when administered intravenously is reduced due to the presence of neutralizing antibodies. In this study, we evaluated the role of exosomes in human umbilical cord-derived mesenchymal stem cells (UC-MSCs) to deliver reovirus to AML cells. We show that UC-MSCs loaded with reovirus can deliver reovirus to tumor cells without cellular contact. We further demonstrate that the exosome inhibitor, GW4869, inhibits the release of exosomes as well as inhibited the transfer of reovirus from UC-MSCs to tumor cells. Mechanistically, we show that exosomes derived from reovirus-infected UC-MSCs (MSCREO-EXOs) have a tumor lysis effect and transmit reovirus to tumor cells mainly through clathrin-mediated endocytosis (CME) and macropinocytosis. In addition, we demonstrate the feasibility of using MSC-derived exosomes (MSC-EXOs) as a reovirus carrier to exert an anti-tumor effect on AML cells. Collectively, our data indicate that UC-MSCs transfer reovirus to AML cells via exosome release and prompt further study of MSC-EXOs as a potential reovirus carrier to treat AML.
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Exosomas , Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Viroterapia Oncolítica , Virus Oncolíticos , Cordón Umbilical , Humanos , Exosomas/metabolismo , Células Madre Mesenquimatosas/virología , Células Madre Mesenquimatosas/metabolismo , Leucemia Mieloide Aguda/terapia , Cordón Umbilical/citología , Virus Oncolíticos/fisiología , Viroterapia Oncolítica/métodos , Línea Celular Tumoral , Reoviridae/fisiología , Compuestos de Anilina/farmacología , Endocitosis , Compuestos de BencilidenoRESUMEN
BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.
Asunto(s)
Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Reoviridae , Reoviridae , Animales , Carpas/genética , Carpas/virología , Carpas/inmunología , Enfermedades de los Peces/virología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/genética , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/genética , Infecciones por Reoviridae/virología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Reoviridae/fisiología , Perfilación de la Expresión Génica , Transcriptoma , Replicación Viral , Regulación de la Expresión GénicaRESUMEN
Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.
Asunto(s)
Aeromonas hydrophila , Carpas , MicroARNs , ARN Mensajero , Reoviridae , Transcriptoma , Animales , Carpas/genética , Carpas/microbiología , Carpas/virología , Carpas/inmunología , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reoviridae/fisiología , Proteómica/métodos , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Enfermedades de los Peces/genética , Perfilación de la Expresión Génica , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/genética , Línea Celular , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/genética , Redes Reguladoras de GenesRESUMEN
The mode and outcome of fish-virus interactions are influenced by many abiotic factors, among which water temperature is especially important in poikilothermic fish. Rare minnow Gobiocypris rarus is a eurythermal small cyprinid fish that is sensitive to infection with genotype II grass carp reovirus (GCRV). HSP70, a conservative and key player in heat shock response, is previously identified as an induced pro-viral factor during GCRV infection in vitro. Here, rare minnow was subjected to heat shock treatment (HST), 1 h treatment at 32 °C followed by reverting to a normal temperature of 24 °C, and subsequently challenged with GCRV-II at a dosage of 1 × LD50. The effect of HST on GCRV virulence in vivo was evaluated by calculating virus-associated mortality and viral load in both dead and survival fish. The results revealed that HST enhanced the mortality of rare minnow infected with GCRV; the fact that viral loads in the tissue samples of HST-treated fish were significantly higher than those in samples of the control group at 6, 8 d p.i. reflected a faster infection process due to HST. Quantitative gene expression analysis was further employed to show that the expression levels of Hsp70 in intestine and liver tissues from the HST group declined faster than muscle tissue after HST. HST W/O GCRV challenge upregulated proinflammatory cytokines such as MyD88 and Nf-κB, which was in consistence with the inflammation observed in histopathological analysis. This study shed light on the complexity of the interaction between fish abiotic and biotic stress response, which suggested that HST, an abiotic stress, could enhance the virulence of GCRV in Gobiocypris rarus that involved modulating the gene expression of host heat shock, as well as a pro-inflammatory response.
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Cyprinidae , Enfermedades de los Peces , Infecciones por Reoviridae , Reoviridae , Animales , Enfermedades de los Peces/virología , Reoviridae/patogenicidad , Reoviridae/genética , Reoviridae/fisiología , Virulencia , Infecciones por Reoviridae/virología , Infecciones por Reoviridae/veterinaria , Cyprinidae/virología , Carga Viral , Carpas/virología , Respuesta al Choque Térmico , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , CalorRESUMEN
Many plant arboviruses are persistently transmitted by piercing-sucking insect vectors. However, it remains largely unknown how conserved insect Toll immune response exerts antiviral activity and how plant viruses antagonize it to facilitate persistent viral transmission. Here, we discover that southern rice black-streaked dwarf virus (SRBSDV), a devastating planthopper-transmitted rice reovirus, activates the upstream Toll receptors expression but suppresses the downstream MyD88-Dorsal-defensin cascade, resulting in the attenuation of insect Toll immune response. Toll pathway-induced the small antibacterial peptide defensin directly interacts with viral major outer capsid protein P10 and thus binds to viral particles, finally blocking effective viral infection in planthopper vector. Furthermore, viral tubular protein P7-1 directly interacts with and promotes RING E3 ubiquitin ligase-mediated ubiquitinated degradation of Toll pathway adaptor protein MyD88 through the 26 proteasome pathway, finally suppressing antiviral defensin production. This virus-mediated attenuation of Toll antiviral immune response to express antiviral defensin ensures persistent virus infection without causing evident fitness costs for the insects. E3 ubiquitin ligase also is directly involved in the assembly of virus-induced tubules constructed by P7-1 to facilitate viral spread in planthopper vector, thereby acting as a pro-viral factor. Together, we uncover a previously unknown mechanism used by plant arboviruses to suppress Toll immune response through the ubiquitinated degradation of the conserved adaptor protein MyD88, thereby facilitating the coexistence of arboviruses with their vectors in nature.
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Arbovirus , Insectos Vectores , Transducción de Señal , Receptores Toll-Like , Animales , Arbovirus/inmunología , Receptores Toll-Like/metabolismo , Insectos Vectores/virología , Insectos Vectores/inmunología , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/inmunología , Reoviridae/fisiología , Reoviridae/inmunología , Hemípteros/virología , Hemípteros/inmunología , Oryza/virología , Oryza/inmunología , Proteínas de Insectos/metabolismo , Inmunidad InnataRESUMEN
In this issue of Cell Host & Microbe, Shang et al. identify murine neuropilin 1 as a host factor that binds reovirus particles, directing cell entry and contributing to viral dissemination and neurovirulence. This study highlights the reovirus model system to investigate host receptors and their significance in viral pathogenesis.
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Neuronas , Neuropilina-1 , Reoviridae , Internalización del Virus , Animales , Ratones , Neuronas/virología , Neuropilina-1/metabolismo , Reoviridae/fisiología , Reoviridae/genética , Reoviridae/patogenicidad , Humanos , Interacciones Huésped-Patógeno , Infecciones por Reoviridae/virología , Receptores Virales/metabolismoRESUMEN
RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs.
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Carpas , ARN Helicasas DEAD-box , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Infecciones por Reoviridae , Reoviridae , Proteínas no Estructurales Virales , Animales , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Carpas/inmunología , Carpas/genética , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Reoviridae/fisiología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Inmunidad Innata/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/inmunología , Regulación de la Expresión Génica/inmunologíaRESUMEN
The grass carp (Ctenopharyngodon idella) constitutes a significant economic resource within the aquaculture sector of our nation, yet it has been chronically afflicted by the Grass Carp Reovirus (GCRV) disease. The complement system, a vital component of fish's innate immunity, plays a crucial role in combating viral infections. This research investigates the potential role of MASP1, a key molecule in the lectin pathway of the complement system, in the GCRV infection in grass carp. An analysis of the molecular characteristics of MASP1 in grass carp revealed that its identity and similarity percentages range from 35.10 to 91.00 % and 35.30-91.00 %, respectively, in comparison to other species. Phylogenetically, MASP1 in C. idella aligns closely with species such as Danio rerio, Cyprinus carpio, and Carassius carassius, exhibiting chromosomal collinearity with the zebrafish. Subsequent tissue analysis in both healthy and GCRV-infected grass carp indicated that MASP1's basal expression was predominantly in the liver. Post-GCRV infection, MASP1 expression in various tissues exhibited temporal variations: peaking in the liver on day 5, spleen on day 7, and kidney on day 14. Furthermore, employing Complement Component 3 (C3) as a benchmark for complement system activation, it was observed that MASP1 could activate and cleave C3 to C3b. MASP1 also demonstrated an inhibitory effect on GCRV replication (compared with the control group, VP2 and VP7 decreased by 6.82-fold and 4.37-fold) and enhanced the expression of antiviral genes, namely IRF3, IRF7 and IFN1 (compared with the control group, increased 2.25-fold, 45.38-fold and 22.37-fold, respectively). In vivo protein injection experiments substantiated MASP1's influence on the relative mRNA expression levels of C3 in various tissues and its protein expression in serum. This study also verified that C3 could modulate the expression of antiviral genes such as IFN1 and IRF3.
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Carpas , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Filogenia , Infecciones por Reoviridae , Reoviridae , Animales , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Carpas/inmunología , Carpas/genética , Reoviridae/fisiología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Inmunidad Innata/genética , Regulación de la Expresión Génica/inmunología , Perfilación de la Expresión Génica/veterinaria , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/genética , Secuencia de Aminoácidos , Alineación de Secuencia/veterinariaRESUMEN
The conversion of Adenosine (A) to Inosine (I), by Adenosine Deaminases Acting on RNA or ADARs, is an essential post-transcriptional modification that contributes to proteome diversity and regulation in metazoans including humans. In addition to its transcriptome-regulating role, ADARs also play a major part in immune response to viral infection, where an interferon response activates interferon-stimulated genes, such as ADARp150, in turn dynamically regulating host-virus interactions. A previous report has shown that infection from reoviruses, despite strong activation of ADARp150, does not influence the editing of some of the major known editing targets, while likely editing others, suggesting a potentially nuanced editing pattern that may depend on different factors. However, the results were based on a handful of selected editing sites and did not cover the entire transcriptome. Thus, to determine whether and how reovirus infection specifically affects host ADAR editing patterns, we analyzed a publicly available deep-sequenced RNA-seq dataset, from murine fibroblasts infected with wild-type and mutant reovirus strains that allowed us to examine changes in editing patterns on a transcriptome-wide scale. To the best of our knowledge, this is the first transcriptome-wide report on host editing changes after reovirus infection. Our results demonstrate that reovirus infection induces unique nuanced editing changes in the host, including introducing sites uniquely edited in infected samples. Genes with edited sites are overrepresented in pathways related to immune regulation, cellular signaling, metabolism, and growth. Moreover, a shift in editing targets has also been observed, where the same genes are edited in infection and control conditions but at different sites, or where the editing rate is increased for some and decreased for other differential targets, supporting the hypothesis of dynamic and condition-specific editing by ADARs.
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Adenosina Desaminasa , Fibroblastos , Inosina , Edición de ARN , Transcriptoma , Animales , Ratones , Fibroblastos/virología , Fibroblastos/metabolismo , Inosina/metabolismo , Inosina/genética , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Adenosina/metabolismo , Adenosina/genética , Infecciones por Reoviridae/virología , Infecciones por Reoviridae/genética , Interacciones Huésped-Patógeno , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reoviridae/genética , Reoviridae/fisiologíaRESUMEN
Nonenveloped viruses employ unique entry mechanisms to breach and infect host cells. Understanding these mechanisms is crucial for developing antiviral strategies. Prevailing perspective suggests that nonenveloped viruses release membrane pore-forming peptides to breach host membranes. However, the precise involvement of the viral capsid in this entry remains elusive. Our study presents direct observations elucidating the dynamically distinctive steps through which metastable reovirus capsids disrupt host lipid membranes as they uncoat into partially hydrophobic intermediate particles. Using both live cells and model membrane systems, our key finding is that reovirus capsids actively deform and permeabilize lipid membranes in a cholesterol-dependent process. Unlike membrane pore-forming peptides, these metastable viral capsids induce more extensive membrane perturbations, including budding, bridging between adjacent membranes, and complete rupture. Notably, cholesterol enhances subviral particle adsorption, resulting in the formation of pores equivalent to the capsid size. This cholesterol dependence is attributed to the lipid condensing effect, particularly prominent at an intermediate cholesterol level. Furthermore, our results reveal a positive correlation between membrane disruption extent and efficiency of viral variants in establishing infection. This study unveils the crucial role of capsid-lipid interaction in nonenveloped virus entry, providing new insights into how cholesterol homeostasis influences virus infection dynamics.
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Cápside , Membrana Celular , Colesterol , Reoviridae , Internalización del Virus , Colesterol/metabolismo , Cápside/metabolismo , Membrana Celular/virología , Membrana Celular/metabolismo , Reoviridae/fisiología , Humanos , Animales , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/químicaRESUMEN
NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKß has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKß, we co-transfected CiNIK-FLAG and CiIKKB-GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKß. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKß was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKß up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKß coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits.
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Secuencia de Aminoácidos , Carpas , Proteínas de Peces , Interleucina-8 , FN-kappa B , Proteínas Serina-Treonina Quinasas , Regulación hacia Arriba , Animales , Carpas/genética , Carpas/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , FN-kappa B/genética , FN-kappa B/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Interleucina-8/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Enfermedades de los Peces/inmunología , Transducción de Señal , Reoviridae/fisiología , Filogenia , Quinasa de Factor Nuclear kappa B , Regulación de la Expresión Génica/inmunología , Poli I-C/farmacología , Lipopolisacáridos/farmacología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Alineación de Secuencia/veterinaria , Inmunidad Innata/genética , Secuencia de Bases , Perfilación de la Expresión Génica/veterinariaRESUMEN
Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.
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Anticuerpos Monoclonales , Anticuerpos Antivirales , Carpas , Enfermedades de los Peces , Infecciones por Reoviridae , Reoviridae , Animales , Carpas/inmunología , Carpas/virología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/virología , Reoviridae/inmunología , Reoviridae/fisiología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Ratones , Humanos , Células HEK293 , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Tropismo Viral , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Ratones Endogámicos BALB C , ChinaRESUMEN
Fluorescent protein (FP) tags are extensively used to visualize and characterize the properties of biomolecular condensates despite a lack of investigation into the effects of these tags on phase separation. Here, we characterized the dynamic properties of µNS, a viral protein hypothesized to undergo phase separation and the main component of mammalian orthoreovirus viral factories. Our interest in the sequence determinants and nucleation process of µNS phase separation led us to compare the size and density of condensates formed by FP::µNS to the untagged protein. We found an FP-dependent increase in droplet size and density, which suggests that FP tags can promote µNS condensation. To further assess the effect of FP tags on µNS droplet formation, we fused FP tags to µNS mutants to show that the tags could variably induce phase separation of otherwise noncondensing proteins. By comparing fluorescent constructs with untagged µNS, we identified mNeonGreen as the least artifactual FP tag that minimally perturbed µNS condensation. These results show that FP tags can promote phase separation and that some tags are more suitable for visualizing and characterizing biomolecular condensates with minimal experimental artifacts.
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Proteínas Luminiscentes , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Virales/metabolismo , Condensados Biomoleculares/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Reoviridae/metabolismo , Reoviridae/fisiologíaRESUMEN
Portunid crabs are distributed worldwide and highly valued in aquaculture. Viral infections are the main limiting factor for the survival of these animals and, consequently, for the success of commercial-scale cultivation. However, there is still a lack of knowledge about the viruses that infect cultured portunid crabs worldwide. Herein, the genome sequence and phylogeny of Callinectes sapidus reovirus 2 (CsRV2) are described, and the discovery of a new bunyavirus in Callinectes danae cultured in southern Brazil is reported. The CsRV2 genome sequence consists of 12 dsRNA segments (20,909 nt) encode 13 proteins. The predicted RNA-dependent RNA polymerase (RdRp) shows a high level of similarity with that of Eriocheir sinensis reovirus 905, suggesting that CsRV2 belongs to the genus Cardoreovirus. The CsRV2 particles are icosahedral, measuring approximately 65 nm in diameter, and exhibit typical non-turreted reovirus morphology. High throughput sequencing data revealed the presence of an additional putative virus genome similar to bunyavirus, called Callinectes danae Portunibunyavirus 1 (CdPBV1). The CdPBV1 genome is tripartite, consisting of 6,654 nt, 3,120 nt and 1,656 nt single-stranded RNA segments that each encode a single protein. Each segment has a high identity with European shore crab virus 1, suggesting that CdPBV1 is a new representative of the family Cruliviridae. The putative spherical particles of CdPBV1 measure â¼120 nm in diameter and present a typical bunyavirus morphology. The results of the histopathological analysis suggest that these new viruses can affect the health and, consequently, the survival of C. danae in captivity. Therefore, the findings reported here should be used to improve prophylactic and pathogen control practices and contribute to the development and optimization of the production of soft-shell crabs on a commercial scale in Brazil.
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Braquiuros , Genoma Viral , Filogenia , Reoviridae , Animales , Braquiuros/virología , Reoviridae/genética , Reoviridae/clasificación , Orthobunyavirus/genética , AcuiculturaRESUMEN
Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type â IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.