A Lipase Isolated from the Silkworm Bombyx mori Shows Antiviral Activity against Nucleopolyhedrovirus

Abstract

A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.Insects exhibit effective immune system measures such as humoral and cellular responses against microbial infection (4). Insect immunity in antibacterial reactions has been the most extensively studied (7,8,13). On the other hand, little is known about insect immunity against viruses (16). Bombyx mori nucleopolyhedrovirus (BmNPV) is a most significant virus in the sericultural industry, often causing severe economic damages. The immune mechanisms of B. mori against this virus remain totally obscure.The infection cycle of BmNPV is mediated by two phenotypically different viral particles: the occlusion-derived virus (ODV) and the budded virus (BV) (9). Occlusion bodies consisting of a crystalline matrix of polyhedron proteins contain ODV particles. When the occlusion bodies are ingested by B. mori larvae, they are dissolved by the alkaline gut juice. The enveloped virions are released and then initiate infection in the midgut columnar epithelial cells. In the case of Autographa californica multicapsid nucleopolyhedrovirus, it was shown that a proportion of the parental virus travels through the midgut epithelial layer, possibly utilizing the plasma membrane reticular system, and enters the hemocoel at the same time, infecting the hemocytes (3). In contrast to ODV, the BV particle consists of a single nucleocapsid surrounded by an envelope acquired as it buds from the plasma membrane of an infected cell and spreads beyond the midgut through the tracheae.Studies on antiviral immunity in insects are still in their infancy, and defense mechanisms at an early stage of viral infection in the alimentary canal remain unknown. The aim of the present study was to determine whether B. mori contains proteins showing anti-BmNPV activity in the gut in order to obtain a clue as to the antiviral mechanisms involved in insect immunity.A protein showing anti-BmNPV was purified through ammonium sulfate fractionation, gel filtration, and reverse-phase high-performance liquid-column chromatography (HPLC) (Fig. 1A). The protein was eluted with 49.5% acetonitrile-0.05% trifluoroacetic acid (TFA) by reverse-phase HPLC. The homogeneity of the antiviral peak fraction was examined both by matrix-as...

Identification of a Soluble NADPH Oxidoreductase (BmNOX) with Antiviral Activites in the Gut Juice ofBombyx mori

“…16) Reports on the presence of proteins with antiviral activity displaying protease and lipase activity are also available. 11,12,16) Though the molecular size of the protein identified in our laboratory falls close to the previously reported antiviral proteins, the establishment of the molecular identity of the protein detected by us as an NADPHoxidoreductase like protein signifies that it is different from the previously reported molecules. Further, the levels of this antiviral protein in the gut juices collected from a selected set of silkworm races (multivoltine and bivoltine) indicates that the races which are tolerant to BmNPV infection had relatively higher quantities this protein in their gut juices (Fig.…”

“…Once the infection breaches the midgut epithelial barrier and propagates from infective foci to the hemocoel, however, there are few mechanisms known to account for the resistance and clearance of infection observed in some virus-host combinations. 10) The literature also suggests that insects have evolved digestive enzymes with antiviral properties, as in the case of BmSP-1, BmSP-2, 11) and Bmlipase-1, 12) that check viral propagation at the initial site of viral infection.…”

Characterization and Identification of Proteolytic Bacteria From the Gut of the Velvetbean Caterpillar (Lepidoptera: Noctuidae)

“…It mainly feeds on mulberry leaves, although tricuspid cudrania leaves can also act as the forage of the larvae (Li 1987 PID/larva) is about one fourth in the tricuspid cudrania leaves group ). In 2003, a study indicated the lipase isolated from silkworm larval alimentary canal shows strong antiviral activity against BmNPV, providing evidence that digestive juice may play an important role during peroral infection with BmNPV (Ponnuvel et al 2003). Therefore, to explore the differences of intestinal bacteria between the BmNPV susceptible and non-susceptible silkworm, we used tricuspid cudrania leaves to feed silkworms in order to construct a BmNPV susceptible model.…”

The Plant Virus Tomato Spotted Wilt Tospovirus Activates the Immune System of Its Main Insect Vector, Frankliniella occidentalis

“…Only two virus-induced molecules, pherokine-2 and -3, were identified in Drosophila after intrathoracic injection of the Drosophila C virus (39). A lipase showing antiviral activity against Bombyx mori polyhedrovirus was purified from the digestive juice of B. mori larvae and detected into the insect midgut, the site of virus entry (37). In addition, it has been shown that Flock house virus is both an initiator and a target of RNA silencing in Drosophila host cells (27) and that several members of the baculovirus family of insect viruses are able to induce apoptosis during infection, resulting in limited viral replication (10).…”