Dergi makalesi Açık Erişim

Chilo iridescent virus encodes two functional metalloproteases

2019    Yesilyurt, Aydin; Muratoglu, Hacer; Demirbag, Zihni; Nalcacioglu, Remziye

The genome of Chilo iridescent virus (CIV) has two open reading frames (ORFs) with matrix metalloprotease (MMP) domains. The protein encoded by ORF 136R contains 178 amino acids with over 40% amino acid sequence identity to hypothetical metalloproteases of other viruses, and the protein 165R contains 264 amino acids with over 40% amino acid sequence identity to metalloproteases of a large group of organisms, primarily including a variety of Drosophila species. These proteins possess conserved zinc-binding motifs in their catalytic domains. In this study, we focused on the functional analysis of these ORFs. They were cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf9 cells with an N-terminal His tag, and purified to homogeneity at 72 hours postinfection using Ni-NTA affinity chromatography. Western blot analyses of purified 136R and 165R proteins with histidine tags resulted in 24- and 34-kDa protein bands, respectively. Biochemical assays with the purified proteins, performed using azocoll and azocasein as substrates, showed that both proteins have protease activity. The enzymatic activities were inhibited by the metalloprotease inhibitor EDTA. Effects of these proteins were also investigated on Galleria mellonella larvae. Insecticidal activity was tested by injecting the larvae with the virus derived from the AcMNPV bacmid carrying 136R or 165R ORFs. The results showed that the baculoviruses harbouring the iridoviral metalloproteases caused early death of the larvae compared to control group. These data suggest that the CIV 136R and 165R ORFs encode functional metalloproteases. This study expands our knowledge about iridoviruses, describes the characterization of CIV matrix metalloproteinases, and might ultimately contribute to the use of this virus as a research tool.