RNAi-mediated viral immunity in insects

The term arbovirus refers to a taxonomically diverse group of mostly RNA viruses that share a similar ecology and maintenance mechanism. The name arbovirus, for arthropod-borne virus, refers to a complex life cycle requiring horizontal transmission by an haematophagous arthropod vector/host between susceptible vertebrate hosts. Although most arboviruses cycle between the insect and small mammals or birds, increasingly frequent crossover events to domesticated animals and humans have resulted in both sporadic epidemics, as well as larger sweeping pandemics.

The economic and health impact of these emerging epidemics has dramatically increased interest into understanding how arboviruses evolve and maintain the ability to cycle between very different hosts. The emphasis of research is now shifting from studying arbovirus infection of human cells, to understanding how the virus behaves within the arthropod vector. Increasing evidence suggests that understanding and controlling the infection in the arthropod before crossover to the mammalian host may be key to combating arboviral infections.

In addition to cell type and tissue specific constraints exerted on the arbovirus by the arthropod host, insects have an active antiviral defense called RNA interference (RNAi) that is able to seek out and destroy viral RNA. The antiviral RNA silencing response of invertebrates is systemic, specific, potent and rapid.Therefore, studying the antiviral RNAi response in the arthropod, such as in the model organism Drosophila melanogaster, could unravel the mechanisms by which insects harbor virusesand may identify new approaches to control virus replication in the vector, reducing by consequence, transmission events.

Our lab focuses on the identification and characterization of the mechanism that mediates the antiviral activity of RNA interference in insects.

Since June 2008, we have been running, among others, the projects outlined below:

1- RNAi is the main antiviral response against negative strand RNA viruses in insects

The question of the immune antiviral response to negative strand RNA viruses in insects has not been addressed until now. Negative strand RNA viruses include some of the most important human pathogenic viruses, such as the hemorrhagic fever viruses Ebola and Lassa (mortality rate up to 80%), the Rabies virus (mortality rate 100%) and the influenza virus. There are also some important human pathogens that are (-) strand RNA viruses and are transmitted by insect vectors to humans. In order to study the role of RNAi in the control of (-) strand RNA viruses in insect hosts, we used the well characterized drosophilamodel and Vesicular Stomatitis Virus (VSV).This work has been carried out in collaboration with the team of Jean Luc Imler, at the Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg.

Together, we showed that VSV does not produce detectable amounts of dsRNA in infected drosophila cells. Nevertheless, we observed a dramatic increase in the VSV titers in RNAi mutant flies, leading to high mortality. Small RNA profiling revealed VSV derived siRNA equally matching both strands of the whole genome, consistent with a production from dsRNA. Our results indicate the RNAi is an efficient host defense mechanism in insects against all type of viruses including (-) strand RNA viruses. In addition, our results suggest that even very low amounts of dsRNA in infected cells can be an efficient trigger for antiviral RNAi.

A paper describing these findings has been published in PNAS.

2- Antiviral RNAi as a key regulator in persistent vs. acute infections

Insect-virus interactions are interesting models to understand the complexity of host-pathogen interactions, since many insects act as vectors for an increasing number of emerging human viral diseases. In this case, the viruses are exposed to different antiviral immune responses of the mammalian and the insect hosts. In fact, many insect viruses develop a persistent infection during which cells are infected and produce viral particles, yet without clear signs of infection. In this context a key question is how viruses establish a persistent infection in insects.

DrosophilaC virus, Flock House virus and DrosophilaX virus are capable of infecting Drosophilaand trigger an effective RNAi response that is essential for the insect to survive viral infection. These RNA viruses are cytopathic for DrosophilaS2 cells even though these cells possess a functional antiviral RNAi machinery. Interestingly, we have generated S2 cell lines that are persistently infected by these viruses and show no obvious signs of infection. These cell lines produce a similar amount of total viable viral particles, compared to acutely infected S2 cells that retain their virulence in adult flies. Altogether, these data suggest that the persistent infection state is mainly controlled by the insect antiviral response.

To gain insight into the mechanism underlying the switch from acute to persistent infection, we analyzed the RNAi-mediated antiviral response in both conditions by deep sequencing. To do so, we were prompted to optimize the isolation of small RNAs from infected cells and infected insects and the subsequent generation of small RNA libraries for deep sequencing. We are among the first labs worldwide performing small RNAs profiles in vivo. In pilot studies, we were able to reassemble the entire viral genome of the infecting virus solely by de novo assembly of the small RNA populations. This validates using deep sequencing to perform viral discovery and to study the interphase virus-RNAi response. We developed our own bioinformatics pipeline with which to analyze results. This pipeline has proven to be faster and more effective than commercially available software tools. We currently collaborate with several teams inside and outside Pasteur on the analysis and exploration of small RNA pathways in different model organisms.

Keywords: antiviral RNAi, arboviruses, drosophila, small RNAs, deep sequencing, innate immunity

Mueller S, Gausson V, Vodovar N, Deddouche S, Troxler L, Perot J, Pfeffer S, Hoffmann JA, Saleh MC, Imler JL. (2010). RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila. Proc Natl Acad Sci U S A. 107(45):19390-5

Saleh MC, Tassetto M, van Rij RP, Goic B, Gausson V, Berry B, Jacquier C, Antoniewski C, Andino R. (2009) Antiviral immunity in Drosophila requires systemic RNA interference spread. Nature.Mar 19;458(7236):346-50.