ORIGINAL ARTICLE Transcriptional analysis of ORF amv133 of Amsacta moorei entomopoxvirus Emine Ozsahin • Kazım Sezen • Zihni Demirbag Received: 10 January 2014 / Accepted: 20 April 2014 Ó Springer-Verlag Wien 2014 Abstract The open reading frame (ORF) amv133 of Amsacta moorei entomopoxvirus, encodes a putative lipase gene. Its temporal expression pattern was characterized by RT-PCR and found to start at 6 h postinfection (h p.i.) and reach a maximum level at 48 h p.i. While the ORF has a late promoter motif, the inhibition of viral DNA synthesis by Ara-C failed to inhibit transcription, but a general inhibitor of protein synthesis prevented its transcription, indicating that amv133 is an intermediate gene. 5 0 -RACE analysis showed that transcription was initiated at position -77 relative to the translational start site. To determine the size of the promoter, several truncations were generated and cloned upstream of the firefly luciferase reporter gene. The resulting constructs were tested in a dual assay. A fragment that contained 115 bp relative to the transcription start site exhibited optimum promoter length. Introduction Poxviruses are a family of large cytoplasmic replicating and complex double-stranded DNA viruses. Unlike viruses that replicate in the nucleus, poxvirus particles carry a number of enzymes and other proteins required to initiate replication in the cytoplasm of infected cells [8, 25]. The family Poxviridae is divided into two subfamilies: the Chordopoxvirinae, consisting of vertebrate viruses (e.g., vaccinia, cowpox, variola and myxoma viruses) and the Entomopoxvirinae, whose members infect insects. The two subfamilies have a number of common morphological, structural and biochemical features. The entomopoxviruses are distinguished by generally being occluded late in the infection cycle in oval-shaped occlusion bodies called spheroids that afford the embedded virions a certain amount of protection against environmental inactivating agents [16, 32]. The order of the ‘‘core genes’’ is not conserved between the chordopoxviruses and the entomo- poxviruses [37]. The Entomopoxvirinae are classified into three genera depending on the insect host range, virion morphology and genome size: Alphaentomopoxvirus, Betaentomopoxvirus and Gammaentomopoxvirus [35]. Amsacta moorei entomopoxvirus (AMEV) is the type member of the genus Betaentomopoxvirus. It infects members of the orders Lepidoptera (months and butterflies) and Orthoptera (grasshoppers) [35] and was initially con- sidered for use as a microbial biological control agent [37]. Also, AMEV may have potential as a protein expression [25, 26] and gene therapy vector [3, 15, 19, 23]. To date, AMEV is the best-studied entomopoxvirus because it is one of the few insect poxviruses that can be readily grown and manipulated in cell culture [7, 19, 20, 33]. IPLB-LD-652 cells from the gypsy moth Lymantria dispar [9, 10] and Ea.BTi (EAA-BTI) cells from the salt marsh caterpillar Estigmene acrea [11, 22] are examples of lepidopteran cell lines that are permissive to AMEV [33]. The genome of AMEV is 232,392 bp long [5] and contains 294 potential ORFs [37, 43]. Some of the AMEV genes such as spheroidin [13, 30], thymidine kinase [12, 21], FALPE [1, 2], NAD ? -dependent DNA ligase [36], DNA topoisomerase [34], superoxide dismutase [6], inhibitor of apoptosis [17, 18], p33 [24], poly (A) polymerase [7], protein kinase [27] and DNA photolyase [29] have been transcriptionally or functionally identified and characterized. The ORF amv133 potentially encodes a putative triac- ylglycerol lipase with homologs in other entomopoxviruses E. Ozsahin  K. Sezen  Z. Demirbag (&) Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey e-mail: zihni@ktu.edu.tr 123 Arch Virol DOI 10.1007/s00705-014-2096-1