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Nora virus is an enteric virus that causes persistent, non-pathological infection in Drosophila melanogaster. It replicates in the fly gut and is transmitted via the fecal-oral route. Nora virus has a single-stranded positive-sense RNA genome, which is translated in four open reading frames. Reading frame three encodes the VP3 protein, the structure and function of which we have investigated in this work. We have shown that VP3 is a trimer that has an α-helical secondary structure, with a functionally important coiled-coil domain. In order to identify the role of VP3 in the Nora virus life cycle, we constructed VP3-mutants using the cDNA clone of the virus. Our results show that VP3 does not have a role in the actual assembly of the virus particles, but virions that lack VP3 or harbor VP3 with a disrupted coiled coil domain are incapable of transmission via the fecal-oral route. Removing the region downstream of the putative coiled coil appears to have an effect on the fitness of the virus but does not hamper its replication or transmission. We also found that the VP3 protein and particularly the coiled coil domain are crucial for the stability of Nora virus virions when exposed to heat or proteases. Hence, we propose that VP3 is imperative to Nora virus virions as it confers stability to the viral capsid.

RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.

Pathogens induce the activation and suppression of several genes, including potent immune response genes in Drosophila melanogaster. Although several different immune response pathways have been shown to play important roles in the defense against viruses, most of these responses are virus-specific and there appears to be no pathway or set of genes that are induced in response to all viruses. We have here identified genes that are differentially expressed in Drosophila melanogaster in response to Nora virus infection. Our results indicate that none of the known immune response pathways are induced following Nora virus infection, but among the upregulated genes we find three members of the same family as CHKov1 and CHKov2 (ref(3)D), which are involved in resistance to the σ virus. Although Nora virus is a known target of the RNAi pathway, none of the genes belonging to this antiviral system were affected. On the contrary, known immunity responders, such as targets of the Toll and Imd pathways, were downregulated. Several heat-shock proteins, which are usually activated in response to external stimuli, are induced during Nora virus infection. Strikingly, several stress response genes, chaperones and genes required for repair were induced, while digestive enzymes and proteins involved in nutrient uptake and transport were shut down.