Spreading dsRNA via receptor mediated endocytosis is required for insect systematic anti-virual immunity

Nature. 2009 Feb 8.

Antiviral immunity in Drosophila requires systemic RNA interference spread.

Saleh MC, Tassetto M, van Rij RP, Goic B, Gausson V, Berry B, Jacquier C, Antoniewski C, Andino R.

[1] Department of Microbiology and Immunology, University of California, San Francisco 94122-2280, USA [2] Present addresses: Institut Pasteur, Viruses and RNA interference, F-75015 Paris, France (M.-C.S.); Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands (R.P.v.R.).

Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 10(5)-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.

PMID: 19204732

Informative quotes:

• Our model states that infected cells release viral dsRNA that is subsequently taken up by uninfected cells through the dsRNA uptake pathway, thereby eliciting an antiviral RNAi response.
• It was previously thought that Drosophila is unable to spread systemically an RNAi response, based on observations that endogenously expressed RNA hairpins do not spread from cell to cell8. However,we demonstrate that, upon virus infection, infected cells spread systemically a silencing signal that elicits protective RNAi-dependent immunity throughout the organism.
• It is remarkable that blocking the spread of the RNAi signal has such a profound effect on antiviral immunity. This suggests that the cell-autonomous RNAi response is insufficient to control a viral infection. In striking parallel to vertebrates, flies also rely on systemic immunity, albeit in this case the virus-specific signal is dsRNA-based.
• These observations provide an insight into the evolutionarily conserved principles of immunity in multicellular organisms, requiring both cell-autonomous responses as well as systemic mechanisms to create pre-existing immunity to protect uninfected cells.

F1000 review:

Adolfo Garcia-Sastre
with Alina Peterson
Department of Microbiology, Mount Sinai School of Medicine, United States of America

The work of Saleh et al. provides an interesting addition to our understanding of antiviral immunity in insects. This study nicely ties in two characterized Drosophila pathways, RNA interference and double-stranded RNA (dsRNA) uptake, to create a more complete picture of the fly immune system. Importantly, it illustrates that in addition to cellular immunity, a more systemic innate immune response -- be it small interfering RNA-based such as in plants and insects or protein-based such as in vertebrates -- is required in order to mount an effective antiviral defense.
The RNA interference pathway has been recognized to be the primary immune response to viral infections in insects. However, up to now, this pathway was thought to function only in a cell-autonomous manner and to not provide systemic protection to the infected animal. The findings of Saleh et al. demonstrate that in Drosophila (analogous to plants) the antiviral siRNA response initiated upon viral infection is propagated within the organism resulting in a creation of an antiviral state in uninfected cells. Saleh and coworkers were able to show that viral titers and lethality from Sindbis and Drosophila C virus (DCV) infections were greatly enhanced in flies lacking a functional dsRNA uptake response, even in the presence of competent siRNA machinery. In addition, the authors were able to demonstrate that in transgenic flies, GFP expression in the head was silenced upon intrathoracic inoculation with Sindbis–GFP, days before any viral RNA could be detected in this organ. Together, the data suggest that the silencing signal was transmitted to regions distal from the site of infection. In addition to presenting a novel component of Drosophila immunity, this work also highlights the challenges of counteracting two distinct immune systems in insect and vertebrate hosts as well as the ability to suppress both siRNA-based and protein-based innate immunity faced by arboviruses. The conclusions of this paper again bring up an important and yet unanswered question as to whether RNA silencing-based immunity exists in vertebrates.