Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens.
ABSTRACT ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.
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ABSTRACT: Larvae of the black cutworm, Agrotis ipsilon Hufnagel, were more susceptible to infection by Agrotis ipsilon multiple nucleopolyhedrovirus (AgipMNPV) after feeding on Herculex ® I, a transgenic corn hybrid expressing Cry1Fa2 compared to larvae fed on isoline corn. Experiments were conducted to investigate the physiological basis for increased susceptibility to virus infection following exposure to Herculex ® I. Midgut pH, gut protease activity and peritrophic matrix structure are important factors for both Bt toxic action and baculovirus infection. No significant treatment differences were found in the pH of anterior midgut, central midgut or posterior midgut in larvae fed Herculex ® I or isoline diets. Analysis of soluble and membrane–associated gut proteinase activities from larvae fed Herculex ® I or isoline diets indicated that membrane–associated aminopeptidase activity and soluble chymotrypsin–like proteinase activity were significantly lower in Herculex ® I –fed larvae compared to isoline–fed larvae. The number and relative molecular masses of soluble chymotrypsin–like proteinases did not differ. An experiment to determine whether chymotrypsin degradation of baculovirus resulted in reduced infection of larvae fed on isoline diet showed that baculoviruses are not susceptible to degradation by chymotrypsin. Analysis of the peritrophic matrices of Herculex ® I –fed larvae and isoline-fed larvae by scanning electron microscopy indicated that Herculex ® I did not result in damage to the peritrophic matrix that could facilitate subsequent baculovirus infection. Further analyses are required to determine whether Bt toxin–induced epithelial cell sloughing enhances subsequent virus infection.
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ABSTRACT: Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.
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ABSTRACT: Alphabaculoviruses of the family Baculoviridae infect the larvae of the Lepidoptera (moths and butterflies) when the occlusion-derived virus (ODV) released from the occlusion body (OB) binds and fuses to the midgut epithelium. Most alphabaculoviruses readily infect only a few species of caterpillars. The ODV contain more than 30 proteins, twelve of which are conserved across the alphabaculoviruses, including the envelope proteins ODV-E56 and ODV-E66. The mechanism of viral fusion and entry, as well as, the ODV envelope proteins implicated in this process, are unknown. A family of ODV envelope per os infectivity factors (PIFs) has been identified, which includes seven proteins that have significant effects on oral infectivity. Here, we assess the potential roles of ODV-E56 and ODV-E66 in oral infectivity. Bioassays showed that ODV-E56-negative virus was significantly less infectious per os, in both ODV and OB. However, the ODV-E56 negative purified ODV exhibited a two-log reduction in oral infectivity compared to ODV-E56 positive virus, whereas the ODV-E56 negative OBs exhibited a five-log reduction in infectivity. This suggests ODV-E56 may function in early interactions within the gut. The ODV-E56 negative viruses exhibited wild-type levels of binding and fusion, but viral DNA was not transcribed. ODV-E56 bound to 97 kDA protein from Heliothis virescens midguts. Thus, ODV-E56 is not essential for cell fusion, but may function in cell signaling and post-fusion events. These results indicate that ODV-E56 is PIF-5. Fifteen H. virescens gut-binding peptides were isolated using a phage display library, and two peptides showed similarity to ODV-E66. One phage peptide, HV1, exhibited strong binding to cryosections of fourth instar H. virescens midguts, and in competition assays with baculovirus resulted in decreased mortality and increased survival time. The homolog AcE66A to the second phage peptide (HV2), exhibited strong binding, but had no effect on mortality in competition assays. ODV-E56 and ODV-E66 are conserved proteins that localize to a very complex virion envelope, and further studies are needed to detail their functions. Increased knowledge of the determinants of virus infection may facilitate further development of these viruses for use as environmentally benign insecticides, as well as, greater understanding of viral mechanisms.