A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus

T Ohkawa, K Majima, S Maeda
1994 Journal of Virology  
Sequence analysis of the BamHI F fragment of the genome of Bombyx mona nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in
more » ... . In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a ,1-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those ofwild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Baculoviruses are characterized by double-stranded circular genomes packaged within rod-shaped enveloped capsids. The family Baculoviridae is composed of three subgroups, nuclear polyhedrosis viruses (NPVs), granulosis viruses, and nonoccluded baculoviruses. NPVs have been used as vectors for the high-level expression of foreign genes (for a review, see reference 22). NPVs and granulosis viruses have also been studied as alternatives to chemical insecticides (for a review, see reference 6). Autographa califomica NPV (AcNPV) is the most wellcharacterized baculovirus, the next most well-characterized being Orgyia pseudotsugata NPV and Bombyx mori NPV (Bm NPV) (see reference 19). More than 60% of the AcNPV genome has been sequenced (13), resulting in the discovery of a number of interesting genes, including UDP-glucosyltransferase (21), apoptosis-blocking protein (2), and ubiquitin (7) (see reference 22). Nucleotide sequence analysis of the Bm NPV genome in our laboratory (5) indicates that AcNPV and BmNPV share about 95% homology in 90% of the sequenced regions (see references 10 and 19). BmNPV and AcNPV are especially useful models for the study of virus host range determinants, since BmNPV and AcNPV have nonoverlapping hosts but very high nucleotide sequence homology. Proteases from a number of viruses related to the papain, trypsin, and aspartate proteinase superfamilies have been identified (for a review, see reference 31). But with the exception of the adenovirus L3 23-kDa proteinase and human immunodeficiency virus type 1 protease, which have been shown to cleave host intermediate filament proteins (1, 27) , viral proteases have been implicated only in the cleavage of polyprotein precursors. Previously described viral proteases show a remarkable specificity for sites where precursor cleavage will generate mature proteins and have not been found to play a role in protein turnover or degradation. Although these viral proteases have catalytic-site residues identical to those of cellular proteases, the overall sequence homologies to cellular proteases are low. Viruses which encode trypsin-like proteases include alphaviruses, flaviviruses, picornaviruses, and a number of plant viruses (for reviews, see references 8 and 14). Viruses with papain-like proteases include alphaviruses, coronaviruses, and tobacco etch virus (for a review, see reference 14). Aspartate proteases have been found in retroviruses (for reviews, see reference 23 and 28). The viral papain-like cysteine protease genes in general show limited sequence similarity only for active-site residues. Furthermore, the spacing between the conserved catalytic residues is different from that found in papain superfamily cysteine proteases. In the AcNPV genome, a partial papainlike sequence has been identified (25) upstream of the AcNPV gp64 gene (34). The deduced amino acid sequence of the incomplete open reading frame showed 32% identity to that of papain and had conserved catalytic residues and cysteine residues putatively involved in the formation of disulfide bridges. However, an increase in cysteine protease activity was not found in AcNPV-infected SF9 cells. Stimulation of acidic cysteine protease activity in BmNPVinfected isolated pupal abdomens of the silkworm, B. mon, has been reported previously (12). It was also reported that cysteine protease activity is stimulated by ecdysone injection into isolated pupal abdomens. The virus-stimulated protease activity was hypothesized to be of host origin, although the chromatographic patterns of activity differed from those of the ecdysone-stimulated activity.
doi:10.1128/jvi.68.10.6619-6625.1994 fatcat:p3625y2eyzg4zcitmngxlhejiy