Weichert W S, Parker J S, Wahid A T, Chang S F, Meier E, Parrish C R
College of Veterinary Medicine, Cornell University, Ithaca, New York, 14853, USA.
Virology. 1998 Oct 10;250(1):106-17. doi: 10.1006/viro.1998.9352.
The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.
对犬细小病毒（CPV）的衣壳进行了蛋白酶敏感性和结构变异分析。从组织培养物中回收的CPV完整（含DNA）颗粒中，VP2天然裂解为VP3发生在序列Arg-Asn-Glu-Arg Ala-Thr内。胰蛋白酶、胰凝乳蛋白酶、菠萝蛋白酶和组织蛋白酶B均可使完整衣壳中>90%的VP2裂解为VP3，但对空衣壳无此作用，且不会进一步消化衣壳。用蛋白酶K、链霉蛋白酶、木瓜蛋白酶或枯草杆菌蛋白酶消化可使VP2裂解为VP3，还会在其他内部位点裂解，导致颗粒解体和蛋白质降解。蛋白酶K或枯草杆菌蛋白酶产生的几种部分消化产物约为31 - 32.5 kDa，表明衣壳蛋白环3内以及其他位点发生了裂解。在pH 5.5或7.5条件下对衣壳进行蛋白酶处理，并未显著改变其消化敏感性。CPV空衣壳的等电点为pH 5.3，完整衣壳的酸性更强0.3个pH单位，但VP2裂解为VP3后，完整衣壳的pI与空衣壳相同。针对各种衣壳蛋白序列的抗体显示，大多数VP2分子的氨基末端位于完整颗粒而非空颗粒的外部，VP1独特序列位于内部，且衣壳可通过加热或尿素处理解体以暴露内部序列。添加到细胞中的衣壳定位于细胞质中的囊泡内，这些囊泡似乎是溶酶体。显微注射的衣壳主要保留在细胞质中，尽管2小时后观察到一小部分位于细胞核中。用[35S]甲硫氨酸标记的CPV衣壳在0℃与细胞结合，细胞升温后，即使长时间孵育，也未观察到VP1或VP2的显著裂解。在用蛋白酶抑制剂存在的情况下用病毒接种细胞，并未显著降低感染率。
