Journal of General Virology (2002), 83, 2857–2867. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF17/18, a homologue of Xestia c-nigrum granulovirus ORF129 Wilfred F. J. IJkel, Els C. Roode, Rob W. Goldbach, Just M. Vlak and Douwe Zuidema Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55 % amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV). Se17/18 was transcribed in cultured S. exigua 301 cells, as a polyadenylated transcript of 1–1 kb. 5«-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon. These sites overlap with a baculovirus consensus early promoter motif. Se17/18 transcripts were detected by Northern blot analysis and RT–PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i. A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E. coli. However, no immunoreactive protein was detected in SeMNPV-infected Se301 cells and S. exigua larvae, neither in concentrated BV and ODV preparations. These observations and the inability to detect a C-terminal GFP-fusion protein of Se17/18 in Se301 cells using a GFP antibody suggest that Se17/18 protein is present, if at all, in spurious amounts. Based on the low homology of the Se17/18 protein to (methyl) transferases its possible involvement in transcription regulation is discussed. Introduction The beet army worm (Spodoptera exigua ; Lepidoptera, Noctuidae) is an agriculturally important pest insect in (sub)tropical regions of the world and in greenhouses (Federici & Maddox, 1996). S. exigua multicapsid nucleopolyhedrovirus (SeMNPV) is a highly pathogenic baculovirus for the beet army worm. SeMNPV differs from many other NPVs in that it is monospecific and highly virulent for S. exigua larvae (Smits, 1987). These biological characteristics make SeMNPV an attractive biological alternative for chemical insecticides. Detailed information on the expression and function of specific SeMNPV genes is important to gain further insight into baculovirus biology. In previous studies, a mutant SeMNPV that lacked virulence in vivo was obtained within the first passage in cell culture (Heldens et al., 1996). On the basis of the entire nucleotide sequence of SeMNPV this mutant contained a contiguous 25 kb deletion encompassing SeMNPV ORFs15 to 41 (IJkel et al., 1999). So far, none of the 27 ORFs deleted has been Author for correspondence : Just Vlak. Fax ›31 317 484820. e-mail just.vlak!wur.nl experimentally shown to be essential for biological activity or virulence in vivo. Alternate cloning of SeMNPV, switching between cell culture and insects, and vice versa, resulted in mutants lacking ORFs15 to 28 (Dai et al., 2000). Of these, SeMNPV ORF17 (Se17) and ORF18 (Se18) were previously characterized as unique to SeMNPV (IJkel et al., 1999). Upon resequencing they appeared to be linked into a single ORF (Se17}18). Surprisingly, Se17}18 has a homologue in Xestia c- nigrum granulovirus (XcGV ; Hayakawa et al., 1999), which is only very distantly related to SeMNPV. By computational analysis it appeared that Se17}18 has a cytoplasmic localiza- tion (Reinhardt & Hubbard, 1998). The function and signifi- cance of Se17}18 and its XcGV homologue are unknown. To gain more insight in the specific role of genes lost in random deletion mutations after propagation in cell culture, the functionality of Se17}18 was investigated by determining its transcriptional and translational activity as well as its sub- cellular localization. Methods + Computer-assisted analysis. Se17}18 (IJkel et al., 1999) and Xc129 (Hayakawa et al., 1999) were analysed using software on the 0001-8517 # 2002 SGM CIFH