Ohkawa T, Majima K, Maeda S
Department of Entomology, University of California, Davis 95616.
J Virol. 1994 Oct;68(10):6619-25. doi: 10.1128/JVI.68.10.6619-6625.1994.
Sequence analysis of the BamHI F fragment of the genome of Bombyx mori 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 BmNPV-CP. 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 beta-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-(4-guanidino)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 of wild-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.
家蚕核型多角体病毒（BmNPV）基因组BamHI F片段的序列分析揭示了一个开放阅读框，其推导的氨基酸序列与木瓜蛋白酶超家族的半胱氨酸蛋白酶具有同源性。推定的半胱氨酸蛋白酶序列（BmNPV-CP）长323个氨基酸，与布氏锥虫的一种半胱氨酸蛋白酶前体具有35%的同一性。在组织蛋白酶B、H、L和S以及木瓜蛋白酶中保守的36个残基中，31个在BmNPV-CP中是相同的。为了确定推定的半胱氨酸蛋白酶的活性和功能，通过蛋白酶基因与β-半乳糖苷酶基因盒的同源重组构建了一个BmNPV突变体（BmCysPD）。与野生型病毒感染的细胞提取物相比，BmCysPD感染的BmN细胞提取物的酸性蛋白酶活性显著降低。半胱氨酸蛋白酶抑制剂E-64[反式环氧琥珀酰亮氨酰胺基-（4-胍基）丁烷]抑制野生型病毒表达的蛋白酶活性。半胱氨酸蛋白酶基因的缺失对BmN细胞中的病毒生长或多角体产生没有显著影响，表明半胱氨酸蛋白酶在体外病毒复制中不是必需的。然而，感染BmCysPD的家蚕幼虫表现出与野生型BmNPV感染的幼虫不同的症状，例如身体降解较少，包括脂肪体细胞，体表颜色变白可能是由于未降解的表皮细胞，以及释放到血淋巴中的多角体数量增加。这是关于（i）一种对一般底物具有活性的病毒编码蛋白酶和（ii）病毒编码蛋白酶可能在感染幼虫的降解中起作用以促进病毒水平传播的证据的首次报道。
