Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provide information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C, and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights, and when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature Drosophila C Virus (DCV) infection causes low mortality and persistent infection, whereas the closely related Cricket Paralysis Virus (CrPV) causes a lethal infection. We show these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA (dsRNA) and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Ago2 and inhibits its activity, without affecting the miRNA-Ago1 mediated silencing. The link between viral RNAi suppressors and the outcome of infection was examined using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a dramatic increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors.

Multicellular organisms evolved sophisticated defense 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 locations1-4. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity5-7. However, the antiviral RNAi defense in flies is thought to be a local, cell-autonomous process, since flies are considered unable to generate a systemic RNAi response8. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway9 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 (DCV) and Sindbis virus. Mortality in dsRNA-uptake defective flies was accompanied by 100-to 105-fold increases in viral titers 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. Strikingly, infection with Sindbis-GFP 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.

SUMMARY The Aedes aegypti mosquito transmits arboviruses including dengue, chikungunya and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short-interfering RNA (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral RNA viruses that are inserted into the mosquito genome and act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of LTR retrotransposons and organize into large loci (>50kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions.

Effective antiviral protection in multicellular organisms relies on both cell autonomous and systemic immunity. Systemic immunity mediates the spread of antiviral signals from infection sites to distant uninfected tissues. In arthropods, RNA interference (RNAi) is responsible for antiviral defense. Here we show that flies have a sophisticated systemic RNAi-based immunity mediated by macrophage-like haemocytes. Haemocytes take up dsRNA from infected cells and, through endogenous transposon reverse transcriptases, produce virus-derived complementary DNAs (vDNA). These vDNAs template de novo synthesis of secondary viral siRNAs (vsRNA) which are secreted in exosome-like vesicles. Strikingly, exosomes containing vsRNAs, purified from haemolymph of infected flies, confers passive protection against virus challenge in naïve animals. Thus, similar to vertebrates, insects use immune cells to generate immunological memory, in the form of stable vDNAs, that generate systemic immunity, which is mediated by the vsRNA-containing exosomes.