RESUMEN
RNA flow between organisms has been documented within and among different kingdoms of life. Recently, we demonstrated horizontal RNA transfer between honeybees involving secretion and ingestion of worker and royal jellies. However, how the jelly facilitates transfer of RNA is still unknown. Here, we show that worker and royal jellies harbor robust RNA-binding activity. We report that a highly abundant jelly component, major royal jelly protein 3 (MRJP-3), acts as an extracellular non-sequence-specific RNA-aggregating factor. Multivalent RNA binding stimulates higher-order assembly of MRJP-3 into extracellular ribonucleoprotein granules that protect RNA from degradation and enhance RNA bioavailability. These findings reveal that honeybees have evolved a secreted dietary RNA-binding factor to concentrate, stabilize, and share RNA among individuals. Our work identifies high-order ribonucleoprotein assemblies with functions outside cells and organisms.
Asunto(s)
Abejas/genética , Ácidos Grasos/genética , Transferencia de Gen Horizontal/genética , Glicoproteínas/genética , Proteínas de Insectos/genética , Animales , Ácidos Grasos/biosíntesis , Transición de Fase , ARN/genética , Transporte de ARN/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.
Asunto(s)
Sistemas CRISPR-Cas , ADN Circular , VIH-1/genética , Provirus/genética , Secuencias Repetidas Terminales , Proteína 9 Asociada a CRISPR , Edición Génica , Regulación Viral de la Expresión Génica , Terapia Genética , Células HEK293 , Infecciones por VIH/virología , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.
Asunto(s)
Abejas/genética , Agentes de Control Biológico , Silenciador del Gen , Interacciones Huésped-Parásitos/inmunología , ARN Bicatenario/administración & dosificación , Varroidae/patogenicidad , Animales , Abejas/parasitología , Northern Blotting , Interacciones Huésped-Parásitos/genética , Interferencia de ARN , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Varroidae/fisiologíaRESUMEN
The IL-60 platform, consisting of a disarmed form of tomato yellow leaf curl virus (TYLCV) and auxiliary components, was previously developed as a nontransgenic universal vector system for gene expression and silencing that can express an entire operon in plants. IL-60 does not allow rolling-circle replication; hence, production of viral single-stranded (ss) DNA progeny is prevented. We used this double-stranded (ds) DNA-restricted platform (uncoupled from the dsDNAâssDNA replication phase of progeny viral DNA) for functional genomics studies of TYLCV. We report that the noncoding 314-bp intergenic region (IR) is the only viral element required for viral dsDNA replication. None of the viral genes are required, suggesting recruitment of host factors that recognize the IR. We further show that IR-carrying reporter genes are also capable of replication but remain confined to the cells into which they were introduced. Only two sense-oriented viral genes (V1 and V2) need to be added to the IR-carrying construct for expression and movement. Hence, any IR-dsDNA construct supplemented with V1 and V2 becomes a replication-competent, mobile and expressing plant plasmid. All viral functions (replication, expression and movement) are determined by the IR and the sense-oriented genes. The complementary-oriented viral genes have auxiliary roles in the late phase of the virus "life cycle". The previously reported involvement of some viral genes in expression and movement is therefore revised.
Asunto(s)
Begomovirus/fisiología , Expresión Génica , Interacciones Huésped-Patógeno , Replicación Viral , Begomovirus/genética , ADN Intergénico , Genoma ViralRESUMEN
Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plant's life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Operón/genética , Pseudomonas fluorescens/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Cromatografía Líquida de Alta Presión , Replicación del ADN/genética , Resistencia a la Enfermedad/inmunología , Genes Bacterianos/genética , Solanum lycopersicum/inmunología , Espectrometría de Masas , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Pirrolnitrina/química , Pirrolnitrina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rhizoctonia/fisiologíaRESUMEN
The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.
Asunto(s)
Abejas/virología , Colapso de Colonias/prevención & control , Dicistroviridae/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Animales , Apicultura/métodos , Abejas/genética , Clima , Colapso de Colonias/virología , Dicistroviridae/patogenicidad , Florida , PennsylvaniaRESUMEN
Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Worker and royal jellies harbor differential naturally occurring RNA populations. Jelly RNAs corresponded to honey bee protein-coding genes, transposable elements, and non-coding RNA, as well as bacteria, fungi, and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Our findings suggest a transmissible RNA pathway, playing a role in social immunity and signaling between members of the hive.
Asunto(s)
Abejas/genética , Interferencia de ARN/fisiología , ARN Bicatenario/genética , Transducción de Señal/genética , Animales , Ácidos Grasos/genética , Ácidos Grasos/fisiología , Transferencia de Gen Horizontal/fisiología , Larva/genética , Larva/metabolismo , Larva/fisiología , ARN Bicatenario/fisiologíaRESUMEN
BACKGROUND: Mosquitoes host and pass on to humans a variety of disease-causing pathogens such as infectious viruses and other parasitic microorganisms. The emergence and spread of insecticide resistance is threatening the effectiveness of current control measures for common mosquito vector borne diseases, such as malaria, dengue and Zika. Therefore, the emerging resistance to the widely used pyrethroid insecticides is an alarming problem for public health. Herein we demonstrated the use of RNA interference (RNAi) to increase susceptibility of adult mosquitoes to a widely used pyrethroid insecticide. METHODS: Experiments were performed on a field-collected pyrethroid resistant strain of Ae. aegypti (Rio de Janeiro; RJ). Larvae from the resistant Ae. aegypti population were soaked with double-stranded RNAs (dsRNAs) that correspond either to voltage-gate sodium channel (VGSC), P-glycoprotein, or P450 detoxification genes and reared to adulthood. Adult mortality rates in the presence of various Deltamethrin pyrethroid concentrations were used to assess mosquito insecticide susceptibility. RESULTS: We characterized the RJ Ae. aegypti strain with regard to its level of resistance to a pyrethroid insecticide and found that it was approximately 6 times more resistant to Deltamethrin compared to the laboratory Rockefeller strain. The RJ strain displayed a higher frequency of Val1016Ile and Phe1534Cys substitutions of the VGSC gene. The resistant strain also displayed a higher basal expression level of VGSC compared to the Rockefeller strain. When dsRNA-treated mosquitoes were subjected to a standard pyrethroid contact bioassay, only dsRNA targeting VGSC increased the adult mortality of the pyrethroid resistant strain. The dsRNA treatment proved effective in increasing adult mosquito susceptibility over a range of pyrethroid concentrations and these results were associated with dsRNA-specific small interfering RNAs in treated adults, and the corresponding specific down regulation of VGSC gene expression level. Finally, we demonstrated that the efficiency of our approach was further improved by 'tiling' along the VGSC gene in order to identify the most potent dsRNA sequences. CONCLUSIONS: These results demonstrate that dsRNA applied to mosquito larvae retains its biological activity into adulthood. Thus, the RNAi system reported here could be a useful approach to control the widespread insecticide resistance in mosquitoes and other insect vectors of human diseases.
Asunto(s)
Aedes/efectos de los fármacos , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Piretrinas/farmacología , Interferencia de ARN , ARN Bicatenario/genética , Canales de Sodio Activados por Voltaje/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Aedes/genética , Animales , Humanos , Larva/efectos de los fármacos , Control de Mosquitos/métodos , Mosquitos Vectores/efectos de los fármacos , ARN Bicatenario/metabolismo , ARN Bicatenario/farmacologíaRESUMEN
Divergence among individuals of the same species may be linked to positional retrotransposition into different loci in different individuals. Here we add to recent reports indicating that individual variance occurs due to the integration of non-retroviral (potyviral) RNAs into the host genome via RNA recombination followed by retrotransposition. We report that in bees (Apis mellifera), approximately 30% of all tested populations carry a segment of a dicistrovirus in their genome and have thus become virus-resistant. Reciprocally, segments of host sequences have been found within defective-interfering-like sequences of a dicistrovirus. Similarly, host sequences were found fused to potyviral sequences, previously described integrated into their host genome. A potential, continuous RNA exchange leading to divergence is discussed.
Asunto(s)
Abejas/genética , Abejas/virología , Genoma de los Insectos/genética , Picornaviridae/genética , Potyvirus/genética , Recombinación Genética , Animales , Secuencia de Bases , Virus Defectuosos/genética , Datos de Secuencia Molecular , Fenotipo , Análisis de Secuencia de ADN , Nicotiana/genética , Nicotiana/virología , Integración Viral/genéticaRESUMEN
We report the isolation, purification, genome-sequencing and characterization of a picorna-like virus from dead bees in Israel. Sequence analysis indicated that IAPV (Israeli acute paralysis virus) is a distinct dicistrovirus. It is most homologous to Kashmir bee virus and acute bee paralysis virus. The virus carries a 9487 nt RNA genome in positive orientation, with two open reading frames separated by an intergenic region, and its coat comprises four major proteins, the sizes of which suggest alternate processing of the polyprotein. IAPV virions also carry shorter, defective-interfering (DI)-like RNAs. Some of these RNAs are recombinants of different segments of IAPV RNA, some are recombinants of IAPV RNA and RNA from another dicistrovirus, and yet others are recombinants of IAPV and non-viral RNAs. In several of the DI-like RNAs, a sense-oriented fragment has recombined with its complement, forming hairpins and stem-loop structures. In previous reports, we have shown that potyviral and IAPV sequences are integrated into the genome of their respective hosts. The dynamics of information exchange between virus and host and the possible resistance-engendering mechanisms are discussed.