Vol.:(0123456789) 1 3 Archives of Virology https://doi.org/10.1007/s00705-018-4108-z ORIGINAL ARTICLE Chilo iridescent virus encodes two functional metalloproteases Aydın Yesilyurt 1  · Hacer Muratoglu 2  · Zihni Demirbag 1  · Remziye Nalcacioglu 1 Received: 27 August 2018 / Accepted: 11 November 2018 © Springer-Verlag GmbH Austria, part of Springer Nature 2018 Abstract 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 purifed to homogeneity at 72 hours postinfection using Ni-NTA afnity chromatography. Western blot analyses of purifed 136R and 165R proteins with histidine tags resulted in 24- and 34-kDa protein bands, respectively. Biochemical assays with the purifed 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. Efects 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. Introduction Matrix metalloproteases (MMPs) are a group of neutral zinc- dependent endopeptidases that contain a binding domain on their active site for metal ions (Zn 2+ and Ca 2+ ) that are required for their enzymatic activity. MMPs are involved in the degradation of both matrix and extracellular matrix proteins [22, 27]. Their activities are regulated by tissue inhibitors of metalloproteases (TIMPs) [31] and inhibited by metal chelators [20]. MMPs play a crucial role in tis- sue remodeling, cell/tissue diferentiation, wound healing, embryonic development, and metamorphosis [27, 28]. They also contribute to some pathological conditions such as bac- terial and viral infections, cardiovascular and neurological diseases, chronic infammation, arthritis, fbrosis, and par- ticularly, cancer [10, 11, 26]. MMPs have been extensively studied in mammals, but they have recently been identifed in other vertebrates and invertebrates as well as in plants [16]. MMPs have also been reported in viruses from families Baculoviridae, Iridoviri- dae, Nudiviridae, Poxviridae (subfamily Entomopoxvirinae) and Ascoviridae. All these virus families are double-stranded DNA viruses with no RNA stage that infect invertebrates. However, little is known about viral MMPs. The only func- tionally studied viral MMPs belong to the Xestia c-nigrum granulovirus [20] and Cydia pomonella granulovirus [17]. Chilo iridescent virus (CIV), also known as invertebrate iridescent virus 6 (IIV-6), belongs to the family Iridoviridae and is the type member of the genus Iridovirus [33]. The CIV genome consists of 212,482 base pairs of linear, dou- ble-stranded DNA [18] with 211 putative protein-encoding open reading frames (ORFs) [9]. Two of these ORFs (136R and 165R) are predicted to have a metalloprotease domain Handling Editor: Chan-Shing Lin. * Remziye Nalcacioglu remziye@ktu.edu.tr 1 Department of Biology, Faculty of Science, Karadeniz Technical University, 61080 Trabzon, Turkey 2 Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080 Trabzon, Turkey