Israeli Acute Paralysis Virus Infection Leads to an Enhanced RNA Interference Response and Not Its Suppression in the Bumblebee Bombus terrestris

Kaat Cappelle, Guy Smagghe, Maarten Dhaenens, Ivan Meeus
2016 Viruses  
RNA interference (RNAi) is the primary antiviral defense system in insects and its importance for pollinator health is indisputable. In this work, we examined the effect of Israeli acute paralysis virus (IAPV) infection on the RNAi process in the bumblebee, Bombus terrestris, and whether the presence of possible functional viral suppressors could alter the potency of the host's immune response. For this, a two-fold approach was used. Through a functional RNAi assay, we observed an enhancement
more » ... the RNAi system after IAPV infection instead of its suppression, despite only minimal upregulation of the genes involved in RNAi. Besides, the presence of the proposed suppressor 1A and the predicted OrfX protein in IAPV could not be confirmed using high definition mass spectrometry. In parallel, when bumblebees were infected with cricket paralysis virus (CrPV), known to encode a suppressor of RNAi, no increase in RNAi efficiency was seen. For both viruses, pre-infection with the one virus lead to a decreased replication of the other virus, indicating a major effect of competition. These results are compelling in the context of Dicistroviridae in multi-virus/multi-host networks as the effect of a viral infection on the RNAi machinery may influence subsequent virus infections. During evolution, viruses have not stood by idly while their hosts developed this RNAi defense mechanism. Viruses known to encode viral suppressors of RNAi (VSRs) have been found in plants [12] and insects [13] [14] [15] [16] [17] [18] . These VSRs are often small proteins which exercise their function through various mechanisms such as dsRNA sequestering and Dcr-2 or Ago-2 binding. Within the Dicistroviridae, a family of positive single-stranded RNA viruses infecting arthropods, a 166 amino acids (AA) long protein, called 1A, has been proven to be a functional VSR in both the drosophila C virus [14] and the cricket paralysis virus (CrPV) [15] . Recently, the presence of a similar 1A protein has been suggested in another member of the Dicistroviridae, Israeli acute paralysis virus (IAPV). The proof of its functionality was based on reduced virus titers after silencing the 1A region, compared to targeting the non-coding 5' internal ribosome entry site (IRES) region [19] , indicating the need of 1A for virulence. This method might be insufficient by itself, as 1A is a post-translation product and confounding effects like dsRNA target accessibility were not considered. Another small open reading frame (ORF), tentatively named orfX (or pog), has been predicted in IAPV and its close relatives but not in CrPV [20, 21] , and the presence of the resulting protein has been confirmed for IAPV in honeybees [22] but not for Solenopsis invicta virus 1 (SINV-1) [23] . As of yet, no functionality has been attributed to this putative 94 AA protein. VSRs can be very potent in the inhibition of the RNAi mechanism and therefore important immunosuppressive virulence factors. The actual virulence of a virus, defined as the relative capacity to cause damage in a host, depends on the balance between the power of the suppressor and the capacity of the RNAi mechanism of the host to limit the replication of the virus and, hence, the production of this inhibitor. The aim of this study was to investigate to what extent IAPV can influence RNAi efficiency, and to which direction this balance will sway, in the bumblebee Bombus terrestris. This virus is known to pose an important health danger to pollinators such as honeybees and bumblebees [24, 25] and is a target for RNAi-based antiviral therapeutics [26] . As an extra control, CrPV, with its known VSR in Drosophila, was included. CrPV has not been reported as a problematic infection of the pollinator community and has a broad host range as it was reported to infect species within the insect orders of Heteroptera, Diptera, Lepidoptera and Hymenoptera, at least in experimental conditions [27] [28] [29] . Within the concept of virus multi-host dynamics [30] , the presence of VSRs can severely impact the virus virulence in different hosts, as the immunosuppressive capacity is dependent on the host immune strategies and their sensitivity towards VSRs. Also within the same host species, VSRs can influence the virulence of co-infecting viruses [31] , an important feature now that multi-virus reports in pollinators are emerging [32, 33] . In order to evaluate the effect of IAPV infection on RNAi efficiency in B. terrestris, we used a dual approach. First, a proteomic analysis was performed to confirm the translation of the VSR 1A and predicted OrfX proteins using the data-independent acquisition method with high definition mass spectrometry (HDMS E ). Second, the RNAi efficiency after IAPV and CrPV infection was determined. An assay was developed in which bumblebees were infected with a fixed amount of viral particles and after an incubation period injected with dsRNA targeting peptidylprolyl isomerase A (ppia), a gene known to remain stable during virus infection [34] . Silencing levels were evaluated using reverse transcription quantitative PCR (RT-qPCR), along with expression levels of the RNAi core genes, dcr-2 and ago-2, and the systemic RNAi genes, ninaC, egghead and sid-1 [11]. Materials and Methods Bumblebee Rearing and Injections All experiments were performed using 5-to-10 day old workers, age fixed within each experiment, collected from Bombus terrestris colonies (Biobest NV, Westerlo, Belgium). Several workers were collected from the colonies and verified to be virus-free by PCR [35] . One or two workers were collected from each colony and randomly distributed over the microcolonies for the experiments. These microcolonies were placed in an incubator at 30 • C, 60% relative humidity and in continuous darkness and fed with sugar water (50% w/v, BIOGLUC ® ; Biobest NV) and gamma-irradiated Viruses 2016, 8, 334 3 of 17
doi:10.3390/v8120334 pmid:27999371 pmcid:PMC5192395 fatcat:s3yc7ffcvnho3omoen2ptsppdu