Viral particles of the endogenous retrovirus ZAM from Drosophila melanogaster use a pre-existing endosome/exosome pathway for transfer to the oocyte

BACKGROUND: Retroviruses have evolved various mechanisms to optimize their transfer to new target cells via late endosomes. Here, we analyzed the transfer of ZAM, a retroelement from Drosophila melanogaster, from ovarian follicle cells to the oocyte at stage 9–10 of oogenesis, when an active yolk transfer is occurring between these two cell types. RESULTS: Combining genetic and microscopic approaches, we show that a functional secretory apparatus is required to tether ZAM to endosomal vesicles and to direct its transport to the apical side of follicle cells. There, ZAM egress requires an intact follicular epithelium communicating with the oocyte. When gap junctions are inhibited or yolk receptors mutated, ZAM particles fail to sort out the follicle cells. CONCLUSION: Overall, our results indicate that retrotransposons do not exclusively perform intracellular replication cycles but may usurp exosomal/endosomal traffic to be routed from one cell to another

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects

Cross-species comparative analysis of Dicer proteins during Sindbis virus infection

In plants and invertebrates RNA silencing is a major defense mechanism against virus infections. The first event in RNA silencing is dicing of long double stranded RNAs into small interfering RNAs (siRNAs). The Dicer proteins involved in this process are phylogenetically conserved and have the same domain organization. Accordingly, the production of viral derived siRNAs has also been observed in the mouse, but only in restricted cell types. To gain insight on this restriction, we compare the dicing activity of human Dicer and fly Dicer-2 in the context of Sindbis virus (SINV) infection. Expression of human Dicer in flies inefficiently rescues the production of viral siRNAs but confers some protection against SINV. Conversely, expression of Dicer-2 in human cells allows the production of viral 21 nt small RNAs. However, this does not confer resistance to viral infection, but on the contrary results in stronger accumulation of viral RNA. We further show that Dicer-2 expression in human cells perturbs interferon (IFN) signaling pathways and antagonizes protein kinase R (PKR)-mediated antiviral immunity. Overall, our data suggest that a functional incompatibility between the Dicer and IFN pathways explains the predominance of the IFN response in mammalian somatic cells

In Drosophila melanogaster the COM Locus Directs the Somatic Silencing of Two Retrotransposons through both Piwi-Dependent and -Independent Pathways

BACKGROUND: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells. PRINCIPAL FINDINGS: Here, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus. CONCLUSION: Our data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host. [Corrigendum]

published erratum2016 Apr 202016 01 21importedErratum for : Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host. [Nucleic Acids Res. 2015

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.A correction has been published: Nucleic Acids Research, Volume 44, Issue 7, 20 April 2016, Pages 3477–3478, https://doi.org/10.1093/nar/gkw04

Viral sensing by RNA helicases

International audienceA key aspect of antiviral immunity is the distinction between "self" and "non-self" components. This distinction can be established through the detection of double-stranded RNA (dsRNA), a common sign of viral infection, by cytosolic RNA helicases. Depending on the organism, two major antiviral pathways can be induced by dsRNA helicases: RNA interference (RNAi) and interferon (IFN) signaling. In the RNAi pathway, dsRNAs are recognized by a Dicer protein, and are then used for the sequence-dependent recognition and subsequent degradation of the complementary viral RNAs. In the IFN signaling pathway, dsRNAs are recognized by a RIG-like receptor (RLR), which induces a signaling cascade in order to induce the expression of IFNs, cytokines and chemokines. In this review, we discuss the RNA features that can be used by the cell to detect a viral infection, the two aforementioned types of helicase-mediated sensing, as well as some viral escape mechanisms developed to avoid recognition

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

Sensing viral RNAs by Dicer/RIG-I like ATPases across species

Induction of antiviral immunity in vertebrates and invertebrates relies on members of the RIG-I-like receptor and Dicer families, respectively. Although these proteins have different size and domain composition, members of both families share a conserved DECH-box helicase domain. This helicase, also known as a duplex RNA activated ATPase, or DRA domain, plays an important role in viral RNA sensing. Crystallographic and electron microscopy studies of the RIG-I and Dicer DRA domains indicate a common structure and that similar conformational changes are induced by dsRNA binding. Genetic and biochemical studies on the function and regulation of DRAs reveal similarities, but also some differences, between viral RNA sensing mechanisms in nematodes, flies and mammals

Sensing Viral Infections in Insects: A Dearth of Pathway Receptors

International audienceInsects, the most diverse group of animals, can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses can be transmitted to humans, while bee and silkworm viruses cause important economic losses. Like all invertebrates, insects rely solely on innate immunity to counter viral infections. Protein-based mechanisms, involving restriction factors and evolutionarily conserved signaling pathways regulating transcription factors of the NF-kB and STAT families, participate in the control of viral infections in insects. In addition, RNA-based responses play a major role in the silencing of viral RNAs. We review here our current state of knowledge on insect antiviral defense mechanisms, which include conserved as well as adaptive, insect-specific strategies. Identification of the innate immunity receptors that sense viral infection in insects remains a major challenge for the field