Summary auto-generated
This study addressed a major limitation of using Bombyx mori nuclear polyhedrosis virus (BmNPV) as a protein expression vector in silkworm larvae: the degradation of expressed foreign proteins during late-stage infection. The researchers identified a 35 kDa cysteine proteinase appearing in the hemolymph of infected larvae, whose N-terminal sequence matched the BmNPV cysteine proteinase gene. They constructed a deletion mutant virus (CPd) lacking this proteinase gene. The CPd mutant showed no detectable cysteine proteinase activity in hemolymph and produced substantially more stable foreign proteins. When firefly luciferase or human growth hormone genes were expressed using CPd, protein levels remained stable through late infection stages, with approximately twice the luciferase concentration and virtually no degradation products compared to wild-type virus-infected larvae. The CPd-infected larvae also showed less tissue destruction and fewer released polyhedra, indicating the proteinase's role in viral pathogenesis. This improved vector system enables efficient production of protein therapeutics at late infection stages and reduces contamination during protein purification.
Key findings
- A viral-encoded cysteine proteinase accumulates in hemolymph during late BmNPV infection and degrades expressed foreign proteins
- A deletion mutant (CPd) lacking the cysteine proteinase gene produces luciferase and human growth hormone with ~2-fold higher yields and minimal degradation compared to wild-type virus
- CPd-infected larvae exhibit reduced tissue destruction and decreased polyhedra release, revealing the proteinase's role in viral-induced tissue degradation and host lysis
- The cysteine proteinase functions in dispersing polyhedra for virus transmission rather than viral replication itself
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Abstract
Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene. No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd. The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter. These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins. A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive.