In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.
Bottom Line: Isoforms of viral structural proteins of highest abundance were localized via 2D-E.The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding.Hemolymph feeding also resulted in the detection of a variety of honeybee proteins.
Affiliation: Crop Research Institute, Prague 6, Czechia.
We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.
No MeSH data available.
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Mentions: The MS/MS results for the ABPV and VdMLV protein clusters reliably indicated the presence of structural capsid/coat proteins only. In the case of the DWV-cluster, the results indicated a 328-kDa polyprotein that contains the complete DWV protein sequence, including undistinguishable structural and non-structural protein regions. Thus, we divided the polypeptide into structural and non-structural regions according to Lanzi et al.26 (Figure Supplement 2). Further investigation into the sequence coverage of the LC-MS/MS peptide results within the DWV-complex indicated that the identified peptides corresponded to three structural virus proteins, VP2, VP1, and VP3. In addition, one peptide, (K)LKTDLMEMVSNPYIR(R), at position 1448–1462 (see pages 254–255 of the Supplementary Information) of the DWV-complex polypeptide aligned with a helicase domain. One amino acid substitution in the peptide indicated 100% identity with the VDV-1 polyprotein (gi/516317330; see Wang et al.27); other non-redundant NCBI (NCBInr) records showed less significant alignments with the helicase peptide sequence. Additionally, evaluation using Scaffold assigned the helicase peptide to VDV-1, and this peptide was not included in the evaluation of DWV and KV. The single-amino-acid substitution G1456V in the DWV-complex helicase appears specific for VDV-1. The LC-MS/MS identified also single-amino-acid substitution A/K/Q (Fig. 3 and page 244 of the Supplementary Information) with two neighboring serines in VP1 at position 777 of the DWV polypeptide. Details of the peptide identifications (see Table Supplement 4) indicated that the A substitution was high-abundance compared with K and Q substitutions. BLAST search showed that alanine (A) at position 777 (position 292 in VP1) is typically present in the entire DWV-complex of available sequences. Computational investigation of the single-aminoacid-substitution A/K/Q within VP1 for kinase phosphorylation sites (see Figure Supplement 4) revealed that serine S-291 had highest score for phosphoglycerate kinase A (PKA); equal score to S-291 showed S-234 (see page 301 of the Supplementary Information). Substitution Q only slightly decreased score (0.77) for PKA compared to A/K score (0.80). On other hand, the Q substitution had higher score (0.65) for DNA-dependent protein kinase (DNAPK) compared to scores of K (0.49) and A (0.51) substitutions. The 2D-E-MS/MS analysis and further alignment enabled the identification of spots corresponding to VP1 and VP3 of the DWV-complex. VDV-1 helicase or A/K/Q substitutions were not observed via the 2D-E-MS/MS approach.
No MeSH data available.