Determination of cellular proteins playing a role in the start of chilo iridescent virus replication

Chilo iridescent virus (CIV), an insect virus belonging to the Iridoviridae family, has a double-stranded DNA genome (212,482 base pairs) and icosahedral morphology. The DNA genome is surrounded by a lipid layer and a protein capsid on top of this layer. They can be enveloped (separation by budding) or naked (separation by lysis) depending on the way they leave the infected cell. CIV has the potential to be a biopesticide because it causes deadly and non-lethal infections in a large number of pests. In addition, the ability of CIV to enter mammalian cells but not replicate in these cells indicates that the virus can be used to transfer genes to human cells. In order to make effective use of CIV scientifically, it is necessary to elucidate the anatomy, replication and molecular mechanisms of the virus. Proteomic studies have shown that CIV has 54 structural proteins. One of these structural proteins is encoded by the 118L open reading chain (ORF), which may be vital to the virus replication cycle. 118L is a gene homologous to all iridoviruses and ascoviruses. Studies with its homologues indicate that 118L may be a member of the entry-fusion complex of CIV and play an important role in virus replication. In line with this information, in this thesis study, the protein/s with which 118L, an envelope protein of CIV, interacts in the host cell of Spodoptera furgiperda (Sf9) were investigated. First, total membrane proteins isolated from Sf9 cells were hybridized with 118L protein. The protein band formed as a result of hybridization was subjected to LC-MS analysis. Analyse results showed that the protein band potentially resembles four proteins in Sf9 cells, namely Eukaryotic translation initiation factor 3 (EuTIF3), Transitional endoplasmic reticulum ATPase (TER94), Eukaryotic translation elongation factor 2 (TEF2) and Calcium-transporting ATPase sarcoplasmic/endoplasmic reticulum (ctATPser). In-silico experiments showed that TER94 protein interacted with 118L at a high rate. This interaction was confirmed by the interaction of TER94 protein produced in the Baculovirus expression vector system and 118L protein produced in the bacterial system in-vitro at pull-down analysis.