Dataset Information

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

ABSTRACT: Background:The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. Methods:In order to approach the natural conditions of infection and possible integration, we generated linear JcDV-gfp based molecules which were transfected into non permissive Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. Results:We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear "scrambled" whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter. Conclusion:Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

SUBMITTER: Rizk F

PROVIDER: S-EPMC5978394 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Linear <i>Lepidopteran ambidensovirus</i> 1 sequences drive random integration of a reporter gene in transfected <i>Spodoptera frugiperda</i> cells.

Rizk Francine F Laverdure Sylvain S d'Alençon Emmanuelle E Bossin Hervé H Dupressoir Thierry T

PeerJ 20180528

<h4>Background</h4>The <i>Lepidopteran ambidensovirus</i> 1 isolated from <i>Junonia coenia</i> (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA.<h4>Methods</h4>In order to approach the natural conditions of infection and possible integration, we generated linear JcDV-<i>gfp</ ...[more]

PMID: 29868273

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Project description:The success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. As orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. Here, we analyzed the early interaction of the Junonia coenia densovirus (JcDV) with the midgut barriers of caterpillars from the pest Spodoptera frugiperda. Using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. We show that the JcDV particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. Proteomic analyses of JcDV binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. In addition, we show that JcDV early infection results in an arrest of N-Acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. Finally, JcDV early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. These dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. A better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests.

Project description:BACKGROUND:A change in the environment may impair development or survival of living organisms leading them to adapt to the change. The resulting adaptation trait may reverse, or become fixed in the population leading to evolution of species. Deciphering the molecular basis of adaptive traits can thus give evolutionary clues. In phytophagous insects, a change in host-plant range can lead to emergence of new species. Among them, Spodoptera frugiperda is a major agricultural lepidopteran pest consisting of two host-plant strains having diverged 3 MA, based on mitochondrial markers. In this paper, we address the role of microRNAs, important gene expression regulators, in response to host-plant change and in adaptive evolution. RESULTS:Using small RNA sequencing, we characterized miRNA repertoires of the corn (C) and rice (R) strains of S. frugiperda, expressed during larval development on two different host-plants, corn and rice, in the frame of reciprocal transplant experiments. We provide evidence for 76 and 68 known miRNAs in C and R strains and 139 and 171 novel miRNAs. Based on read counts analysis, 34 of the microRNAs were differentially expressed in the C strain larvae fed on rice as compared to the C strain larvae fed on corn. Twenty one were differentially expressed on rice compared to corn in R strain. Nine were differentially expressed in the R strain compared to C strain when reared on corn. A similar ratio of microRNAs was differentially expressed between strains on rice. We could validate experimentally by QPCR, variation in expression of the most differentially expressed candidates. We used bioinformatics methods to determine the target mRNAs of known microRNAs. Comparison with the mRNA expression profile during similar reciprocal transplant experiment revealed potential mRNA targets of these host-plant regulated miRNAs. CONCLUSIONS:In the current study, we performed the first systematic analysis of miRNAs in Lepidopteran pests feeding on host-plants. We identified a set of the differentially expressed miRNAs that respond to the plant diet, or differ constitutively between the two host plant strains. Among the latter, the ones that are also deregulated in response to host-plant are molecular candidates underlying a complex adaptive trait.

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Dataset Information

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

Publications

Linear <i>Lepidopteran ambidensovirus</i> 1 sequences drive random integration of a reporter gene in transfected <i>Spodoptera frugiperda</i> cells.

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