Biomedical subjects

Carine Meignin

Publications and source records attributed to Carine Meignin.

5 recordsLinked to original sources

PubMed2026

Field-isolate recombinant tick-borne encephalitis viruses define reporter-stability guidelines for antiviral testing in flaviviruses.

As arthropod-borne viruses continue to threaten populations globally, there is a pressing need for experimental systems that enable rapid antiviral discovery. Reverse-genetics platforms producing recombinant reporter orthoflaviviruses have been developed to address this gap. Here, we present two new recombinant tick-borne encephalitis viruses (TBEVrec) generated on a European-subtype Haselmühl Tiho1 isolate backbone. A reporter gene, either eGFP or Nluc, was inserted in the capsid-coding region of the genome downstream of the capsid RNA regulatory signal and separated from the complete viral polyprotein by a 2A self-cleaving peptide. TBEVrec was better rescued using the circular polymerase extension reaction (CPER) than with the infectious subgenomic amplicon (ISA) method. TBEVrec replicated efficiently in relevant human cell lines, with comparable replication to wild-type TBEV in a neuronal cell line and moderately reduced titers and RNA levels in immune-derived cell lines. Using either eGFP or Nluc, we illustrate how TBEVrec enabled high-content RNAi screening, highlighting Nucleolin and PRKD1 as potential TBEV host factors, and drug testing on a benchtop plate reader. Nanopore sequencing of the eGFP insert revealed that the reporter is excised without affecting flanking regions. Comparative analysis of eGFP and Nluc further shows that this instability is time- and cell type-dependent, and that Nluc is comparatively more stable. From these observations, we outline safeguards and design principles that are broadly applicable both to the rescue of existing constructs and to the design of future recombinant reporter virus platforms.

CPER↗

PubMed2025

Argonaute 2 targets viral transcripts but not genomes of RNA viruses during antiviral RNA interference in Drosophila.

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we show that AGO2 is associated with ribosomes in control and virus infected cells. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes.

Animals↗

PubMed2004

Intercellular communication between germ line and somatic line is utilized to control the transcription of ZAM, an endogenous retrovirus from Drosophila melanogaster.

ZAM is an long terminal repeat (LTR) retrotransposon from Drosophila melanogaster that bears striking resemblance to the vertebrate retroviruses, in their structure and replication cycle. This element transposes via an RNA intermediate and its reverse transcription, and ultimately inserts copies within the germ line. In this paper, we show that intercellular communication established between the germ line cells and the somatic follicle cells is used to initiate the replication cycle of ZAM. ZAM has been shown to be transcribed in the follicle cells located at the posterior pole of the oocyte. Here, we determine the cis-regulatory elements necessary for its somatic expression, and show that they respond to the EGF-receptor signaling pathway and its activation by the ligand Gurken emitted by the germ line. We further show that the ETS-transcription factor Pointed2 acting downstream of this pathway acts as a trans-regulatory factor and targets a specific cis-regulatory binding site located within the ZAM LTR. Our data give an insight into the molecular mechanism for how intercellular communications between germ cells and somatic cells may be used by endogenous retroviruses to control their replication, and thereby specify their intrinsic and highly restricted expression in the reproductive apparatus.

Animals↗

PubMed2003

COM, a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster.

ZAM and Idefix are two endogenous retroviruses whose expression is tightly controlled in Drosophila melanogaster. However, a line exists in which this control has been perturbed, resulting in a high mobilization rate for both retroviruses. This line is called the U (unstable) line as opposed to the other S (stable) lines. In the process of analyzing this control and tracing the genetic determinant involved, we found that ZAM and Idefix expression responded to two types of controls: one restricting their expression to specific somatic cells in the ovaries and the other silencing their expression in S lines but permitting it in U lines. While studying this second control in the U or S backgrounds, we found that the heterochromatic locus 20A2-3 on the X chromosome, previously implicated in the regulation of a third retroelement, gypsy, also controlled both ZAM and Idefix. We report here that genetic determinants necessary for endogenous retrovirus silencing occur at the 20A2-3 locus, which we call COM, for centre organisateur de mobilisation. We propose that if this point of control becomes mutated during the life of the fly, it may trigger processes reactivating dormant endogenous retroviruses and thus bring about sudden bursts of mobilization.

Animals↗

PubMed2003

The 5' untranslated region and Gag product of Idefix, a long terminal repeat-retrotransposon from Drosophila melanogaster, act together to initiate a switch between translated and untranslated states of the genomic mRNA.

Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5' untranslated region (5'UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5'UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5'UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.

5' Untranslated Regions↗