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We identified a gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) that encodes a small cysteine-rich polypeptide which has size and sequence similarity to omega-conotoxins, a class of calcium ion (Ca2") channel inhibitors, found in the venom of cone snails. Transcriptional analysis indicated that the 159-bp open reading frame, which we named cdl, and a downstream 984-bp open reading frame are transcribed as a single 1.3-kb bicistronic late RNA. The mature ctl gene product was identified as a small secreted protein by high-pressure liquid chromatography fractionation of extraceliular fluid. Viruses with a site-specific deletion in ctl appeared normal with regard to the kinetics and virulence of infection, both in vitro and in vivo. Although we studied the behavior of wild-type and mutant virus-infected insects in some detail, a biological role for ctl in AcMNPV infection remains to be established. * Corresponding author. that the gene product is a small, secreted peptide. The gene is not required for viral infection or replication in vitro or in vivo. We did not observe any difference in the behavior or development of wild-type (wt) or mutant virus-infected insects, and the biological function of ctl remains unknown. MATERIALS AND METHODS Cells and virus. AcMNPV Li (24) was propagated in Spodoptera frugiperda IPLB-SF21 cells (51) as previously described (29). The cells were maintained at 27°C in TC100 medium (GIBCO/BRL, Grand Island, N.Y.) supplemented with 10% fetal bovine serum and 0.26% tryptose broth. RNA isolation. Total RNA was isolated by the guanidinium isothiocyanate method (14) from wt-or AcAINPV-infected SF21 cells. Virus (20 PFU per cell) was allowed to adsorb to cells (107/100-mm plate) for 1 h at room temperature before

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Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (ihe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects ofjhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.