Abstract
Insects have evolved a wide range of strategies to combat invading pathogens, including viruses. Genes that encode proteins involved in immune responses often evolve under positive selection due to their co-evolution with pathogens. Insect antiviral defense includes the RNA interference (RNAi) mechanism, which is triggered by recognition of non-self, virally produced, double-stranded RNAs. Indeed, insect RNAi genes (e.g., dicer and argonaute-2) are under high selective pressure. Honey bees (Apis mellifera) are eusocial insects that respond to viral infections via both sequence specific RNAi and a non-sequence specific dsRNA triggered pathway, which is less well-characterized. A transcriptome-level study of virus-infected and/or dsRNA-treated honey bees revealed increased expression of a novel antiviral gene, GenBank: MF116383, and in vivo experiments confirmed its antiviral function. Due to in silico annotation and sequence similarity, MF116383 was originally annotated as a probable cyclin-dependent serine/threonine-protein kinase. In this study, we confirmed that MF116383 limits virus infection, and carried out further bioinformatic and phylogenetic analyses to better characterize this important gene-which we renamed bee antiviral protein-1 (bap1). Phylogenetic analysis revealed that bap1 is taxonomically restricted to Hymenoptera and Blatella germanica (the German cockroach) and that the majority of bap1 amino acids are evolving under neutral selection. This is in-line with the results from structural prediction tools that indicate Bap1 is a highly disordered protein, which likely has relaxed structural constraints. Assessment of honey bee gene expression using a weighted gene correlation network analysis revealed that bap1 expression was highly correlated with several immune genes-most notably argonaute-2. The coexpression of bap1 and argonaute-2 was confirmed in an independent dataset that accounted for the effect of virus abundance. Together, these data demonstrate that bap1 is a taxonomically restricted, rapidly evolving antiviral immune gene. Future work will determine the role of bap1 in limiting replication of other viruses and examine the signal cascade responsible for regulating the expression of bap1 and other honey bee antiviral defense genes, including coexpressed ago-2, and determine whether the virus limiting function of bap1 acts in parallel or in tandem with RNAi.
Highlights
Conflict is the engine of evolution by natural selection and ecological diversification
Additional advantages of using Sindbis virus (SINV) for in vivo experiments include: lack of confounding infection with this virus, facile virus purification from cultured cells, quantification of virions via plaque assay, lack of viral encoded RNA interference (RNAi) suppressor proteins, and the ability to generate virus from a cDNA cloned construct that includes a carboxy-terminal green fluorescent protein (GFP) tag that facilities virus tracking via microscopy and assessment of virus abundance at the protein level via Western blot analyses [33, 36]
It is important to investigate host-virus specific interactions using a panel of viruses, including both model viruses and naturally infecting honey bee viruses, this was beyond the scope of this study
Summary
Conflict is the engine of evolution by natural selection and ecological diversification. The recognition of PAMPs by PRRs activates immune cascades—such as the Toll (NF-kB/Dorsal), Imd (NF-kB/Relish), and Jak/STAT pathways—which regulate the production and secretion of soluble antimicrobial peptides (AMPs) into the hemolymph [reviewed in [4,5,6, 10,11,12,13]]. These pathways are involved in insect antiviral immunity, the primary antiviral immune response is the RNA interference (RNAi) response [reviewed in [5]]. The RISC surveils the cell for complementary target RNAs, including viral genomes and transcripts, that are recognized, bound, and cleaved—which in turn limits virus infections [14,15,16,17,18,19] [reviewed in [4, 5, 20]]
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