ABSTRACT: Deformed wing virus (DWV) is a major bee pathogen that is actively transmitted by the parasitic mite Varroa destructor and plays a primary role in overwinter colony losses of Apis mellifera. Despite of the intense investigation driven by the unique environmental and economic importance of this essential pollinator species, the mechanisms underlying honeybee-DWV interactions are still poorly understood. Here we report on a group of honeybee proteins that were selectively immunoprecipitated by an antibody directed against DWV particles and present a functional analysis of a major component of this set of potential DWV interactors, which was identified as an arginine kinase (ArgK). ArgK RNA levels positively correlated with DWV load in field collected larvae and honeybees and significantly increased upon DWV injection in controlled laboratory conditions, indicating that the ArgK gene was upregulated by DWV infection. RNAi experiments performed in vitro to target ArgK gene expression resulted in reduced DWV viral load, thus demonstrating that ArgK function is required for DWV replication. Taken together, these data indicate that successful DWV infection relies on transcriptional regulation and active recruitment of host ArgK by this viral pathogen. Therefore, our findings open novel perspectives on the potential control of DWV pathogenicity by targeting its interaction with specific host proteins.
Immunoprecipitated samples in quadruplicate were separated in a precast polyacrylamide gradient gel,run in MES buffer for 25 min at 200 V and the proteins were stained with the Imperial Coomassie stain. Each lane was divided into 6 bands, 48 bands in total, which where destained with 50mM ammonium bicarbonate and acetonitrile (ACN) 1:1 treated for cysteine reduction (10mM dithiothreitol at 56C for 45min) and alkylation (55mM iodoacetamide at RT for 30min in the dark), and for enzymatic digestion with 12.5ng ul trypsin at 37C overnight. The resulting peptide mixture was analyzed by LC MS MS using an Ultimate 3000 HPLC coupled with an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, CA, United States). Peptides were desalted on a trap column and separated on a 19 cm long silica capillary, packed in house with a C18, 1.9um, 100 A resin. The analytical column was encased by a column oven (Sonation; 40C during data acquisition) and attached to a nanospray flex ion source. Peptides were separated on the analytical column by running a 95min gradient of buffer A (5 percent ACN, and 0.1 percent formic acid) and buffer B (95 percent acetonitrile and 0.1 percent formic acid), at a flow rate of 250nl min. The mass spectrometer operated in positive ion mode and full MS was acquired in the Orbitrap in the mass over charge 350 1550 scan range at 120K resolution. Data dependent acquisition was performed in top speed mode (3 s long maximum total cycle): the most intense precursors were selected through a monoisotopic precursor selection (MIPS) filter and with charge greater than 1, quadrupole isolated (1.6 mass over charge width) and fragmented by 30 percent higher energy collision dissociation. Product ion spectra were acquired in the ion trap with a rapid scan rate.
Peptide spectra were analyzed with Proteome Discoverer 2.4 by the search engine Sequest HT using a mixed database containing Apis mellifera (reviewed and unreviewed 20.818 sequences; release 26 01 2022), homo sapiens (42.253 sequences, v2017 10 25) and Oryctolagus cuniculus (892 sequences, release 07 03 2022), and Deformed wing virus (1067 sequences, release 26 01 2022), all downloaded from Uniprot.
Precursor and fragment mass tolerance were set to 15 ppm and 0.6 Da, respectively. Cysteine carbamydomethylation was set as static modification, while methionine oxidation and N-acetylation on protein terminus were set as variable modifications. Specific trypsin cleavages with maximum two miscleavages were admitted. Spectral matches were filtered at 1 percent false discovery rate (FDR), based on q values, and target decoy approach, using the Percolator node. Only master proteins were taken into account.
Quantification was based on precursor intensity of unique and razor peptides.
The Perseus software (1.6.15) was used to perform statistical analyses and evaluate differential protein expression. After contaminant removal (rabbit and human proteins), the abundance values were log2 transformed and filtered for containing at least four valid values in at least one group. Missing values were imputed with the constant value 13. A Students t-test was performed applying 250 randomizations, setting S0=2 and 0.01 FDR, and used to draw a Volcano plot to evaluate significantly different IP over IgG ratios.
Marialuisa Casella 