Functional and structural analysis of amsacta moorei entomopoxvirus putative glycosyl transferase gene (AMV248)

Amsacta moorei entomopoxvirus (AMEV) is an important insect virus which has potential usage as gene expression and therapy vectors and biopesticide. Based on DNA sequencing and bioinformatics analysis, it was suggested that amv248 encodes a putative glycosyltransferase. This suggests that amv248 encodes a structural protein which may play a role at host specificity of the virus and essential for initiation of virus replication. In this thesis, it was aimed to identify the function and structure of amv248 gene. Within the scope of these studies, it was concluded that transcription of amv248 started between 3 and 6 hours post infection, and it belongs to intermediate class of virus gene expression; the 5' and 3' untranslated regions of gene were determined as 126 bases and between 50-84 bases, respectively. The 3D structure of the protein was generated by bioinformatics analysis, and putative host cell binding aminoacids were determined. A 30 kDa recombinant protein of the gene was expressed and shown that it binds to heparine. Specific antibody against protein was produced and virus was immunoneutralized using this antibody. Also it was shown that virus binds to a cellular matrix compound, heparine, viral infection was inhibited by glycoseaminoglycans (GAGs) and viral infection reduced after digestion of GAGs from cell surface. Finally, amv248 knocked-out virus was constructed, and while no differences was found between control and recombinant virus regarding virus replication, infection, protein profile and progeny virus yield; significant change was detected at transcriptional level. In conclusion, important findings regarding to virus-host cell attachment and infection mechanism were determined in this study.