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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- "Second, baculoviruses may exit the midgut using tracheal elements; thus, basal lamina surrounding midgut-associated tracheae in orally infected T. ni with viruses expressing AcFGF was examined by transmission electron microscopy. In the presence of AcFGF, the basal lamina of tracheal cells associated with the midgut was disorganized; instead of a thin uniform sheath surrounding the cells, it appeared fragmented (Means and Passarelli, 2010) and reminiscent of that observed when larvae were infected with viruses expressing basal laminae degrading enzymes (Tang et al., 2007). Analysis with a virus lacking Acfgf showed some rearrangement of tracheal cell basal lamina but not as drastically as with the virus carrying Acfgf. "
ABSTRACT: The mechanisms used by baculoviruses to exit the midgut and cause systemic infection of their insect hosts have been debated for decades. After being ingested, baculoviruses reach the midgut, where several host barriers need to be overcome in order to establish successful infection. One of these barriers is the basal lamina, a presumably virus-impermeable extracellular layer secreted by the epithelial cells lining the midgut and trachea. This review discusses new evidence that demonstrates how these viruses breach the basal lamina and establish efficient systemic infections. The biochemical mechanisms involved in dismantling basal lamina during baculovirus infection may also provide new insights into the process of basal lamina remodeling in invertebrate and vertebrate animals.Virology 02/2011; 411(2):383-92. DOI:10.1016/j.virol.2011.01.009 · 3.32 Impact Factor
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- "In invertebrates, the presence of antigens and the appearance of tissue damage results in the deposition of melanin around the damaged tissue or antigen as well as sclerotization of the cuticle . Melanization of the cuticle and tissue damage, including rupture of the intestine and fragmentation of the fat tissue has been previously shown in larvae of H. virescens infected with a recombinant AcMNPV containing the ScathL gene [38,72,73], suggesting that ScathL was able to cause tissue fragmentation prior to insect death and activate the cascade triggered by serine proteases leading to conversion of pro-phenoloxidase in its active form phenoloxidase. However, Li et al.  have shown that the cystein protease activity of purified ScathL was not able to activate pro-phenoloxidase to phenoloxidase in vitro and the phenoloxidase activity in the hemolymph of H. virescens larvae was not altered by a recombinant AcMNPV containing the ScathL gene under the baculovirus basic p6.9 promoter (AcMLF9.ScathL). "
ABSTRACT: Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricultural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed, and their insecticidal properties analysed against Spodoptera frugiperda larvae. Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.Virology Journal 06/2010; 7(1):143. DOI:10.1186/1743-422X-7-143 · 2.18 Impact Factor
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- "Interestingly, it has been shown that a recombinant baculovirus expressing the S. peregrina cathepsin L (SCathL) induces melanization of larvae (Harrison and Bonning, 2001). In this case, melanization was not directly activated by SCathL but was postulated to be induced by the SCathL-dependent degradation of the basement membrane and recruitment of hemocytes at the sites of injury, because damaged tissues are most probably recognized as non-self (Tang et al., 2007; Li et al., 2008). "
ABSTRACT: Parasites have evolved different virulence strategies to manipulate host physiological functions. The parasitoid wasp Cotesia congregata induces developmental arrest and immune suppression of its Lepidopteran host Manduca sexta. In this interaction, a symbiotic virus (C. congregata Bracovirus, CcBV) associated with the wasp is essential for parasitism success. The virus is injected into the host with wasp eggs and virus genes are expressed in host tissues. Among potential CcBV virulence genes, cystatins, which are tight binding inhibitors of C1A cysteine proteases, are suspected to play an important role in the interaction owing to their high level of expression. So far, however, potential in vivo targets in M. sexta are unknown. Here, we characterized for the first time four M. sexta C1A cysteine proteases corresponding to cathepsin L and cathepsin B and two different '26-29 kDa' cysteine proteases (MsCath1 and MsCath2). Our analyses revealed that MsCath1 and MsCath2 are transcriptionally downregulated in the course of parasitism. Moreover, viral Cystatin1 and MsCath1 co-localize in the plasma following parasitism, strongly suggesting that they interact. We also show that parasitism induces a general increase of cysteine protease activity which is later controlled. The potential involvement of cysteine proteases in defense against parasitoids is discussed.Biological Chemistry 05/2009; 390(5-6):493-502. DOI:10.1515/BC.2009.061 · 3.27 Impact Factor