Cloning and characterization of a Dim1-like mitosis gene of Spodoptera frugiperda cells (Sf9) induced by Autographa californica multiple nucleopolyhedrovirus Mohammad Mehrabadi a,b , Mazhar Hussain a , Sassan Asgari a,⇑ a School of Biological Sciences, The University of Queensland, St. Lucia, QLD 4072, Australia b Department of Plant Protection, University of Tehran, Karaj 31584, Iran article info Article history: Received 28 January 2013 Accepted 18 February 2013 Available online 7 March 2013 Keywords: Dim1-like gene Sf9 Cell proliferation Virus–host interaction AcMNPV abstract Dim1 proteins are evolutionarily highly conserved throughout the eukaryotes and are present in numer- ous species. These proteins are essential for mitosis and pre-mRNA splicing. In this study, the full-length cDNA of Dim1-like gene from Spodoptera frugiperda cells (Sf9) was obtained. S. frugiperda Dim1 (SfDim1) cDNA is comprised of 975 bp including a 429 bp open reading frame (ORF), 225 bp 5 0 untranslated region (5 0 UTR), and 321 bp 3 0 untranslated region (3 0 UTR) with a poly A tail. The predicted polypeptide encoded by this gene is 142 aa with a molecular weight of 16.76 kDa and a PI of 5.53. Sequence alignment and phylogenetic analysis showed high similarities with Dim1 of other species. The evolutionary conserved site of Dim1 proteins ( 35 Asp–Pro–Thr–Cys 38 ) is also present in SfDim1. Silencing of SfDim1 gene decreased cell proliferation at 72 h post-treatment in comparison to mock and control transfected cells. Using RT-PCR, we found relatively higher SfDim1 transcript levels following Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection compared to mock-infected cells from 4 h post- infection (hpi) up until 24 hpi. The expression level diminished dramatically at 36 hpi up to 120 hpi with no expression detected at 144 hpi. Silencing of SfDim1 resulted in lower levels of virus DNA production in comparison to mock-infected cells, which suggested that SfDim1 might benefit the virus and facilitate viral replication. Overall, the results showed that SfDim1 protein is involved in cell proliferation as well as cell–virus interaction. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Dim1 (defective entry into mitosis) is a small protein of 15 kDa that was initially identified in Schizosaccharomyces pombe and shown to be essential both for cell cycle progression as well as for chromosome segregation in mitosis (Berry and Gould, 1997). Dim1 proteins are evolutionarily highly conserved throughout the eukaryotes and are present in numerous species with about 79% sequence identity throughout the entire length of 142 amino acids (Reuter et al., 1999; Zhang et al., 1999). Dim1 deletion from S. pombe genome led to a lethal G2 arrest phenotype which was rescued by overexpression of the homologous mouse Dim1. In con- trast, a Dim1-35 temperature sensitive mutant entered mitosis but showed a loss of viability during this process (Berry and Gould, 1997). Taken together, these findings clearly showed the vital func- tion of Dim1 for entry into mitosis and cell cycle progression. In an effort to characterize the protein composition of Saccharo- myces cerevisiae (U4/U6.U5) tri-snRNP, an orthologue of Dim1 (Dib1) was found to be associated with pre-mRNA splicing sug- gesting that Dib1 might function in pre-mRNA splicing and that its role in cell cycle progression could be indirect (Gottschalk et al., 1999; Stevens and Abelson, 1999). In subsequent studies for characterization of Dim1 function, Dim1 protein was co-puri- fied with the U4/U6.U5 tri-snRNP component Prp1 and was shown to be required for efficient splicing of Lid1 pre-mRNA since S. pom- be cells lacking Dim1 protein or S. cerevisiae cells lacking Dib1 (orthologue of Dim1) failed to splice pre-mRNA in vivo (Carnahan et al., 2005). These findings indicated that Dim1 is implicated in not only cell cycle progression but also in a more specific molecular process such as pre-mRNA splicing. Baculoviruses are a family of large rod-shaped viruses that in- fect arthropods (specially insects) and have double-stranded DNA circular genomes ranging from 80 to 180 kb in size. The most stud- ied baculovirus is Autographa californica multicapsid nucleopolyhe- drovirus (AcMNPV), the type species of Nucleopolyhedrovirus (NPV) of Baculoviridae family, which was originally isolated from a lepi- dopteran insect and contains a 134-kbp genome with 154 open reading frames (ORFs) (Ayres et al., 1994; Jehle et al., 2006). Com- parison of different genomes of baculoviruses has revealed the 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.02.005 ⇑ Corresponding author. Fax: +61 7 3365 1655. E-mail address: s.asgari@uq.edu.au (S. Asgari). Journal of Invertebrate Pathology 113 (2013) 152–159 Contents lists available at SciVerse ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip