Assaying for structural variation in the parvovirus capsid and its role in infection
Assaying for structural variation in the parvovirus capsid and its role in infection
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DOI：
10.1006/viro.1998.9352
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发表时间：
1998-10-10
期刊：
影响因子：
3.7
通讯作者：
Parrish, CR
中科院分区：
文献类型：
--
作者：
Weichert, WS;Parker, JSL;Parrish, CR
对犬细小病毒（CPV）衣壳蛋白酶敏感性和结构变异进行了测定。在从组织培养物回收的CPV全（含DNA）颗粒中，VP 2至VP 3的自然切割发生在序列Arg-Asn-Glu-Arg向下箭头Ala-Thr内。胰蛋白酶、胰凝乳蛋白酶、菠萝蛋白酶和组织蛋白酶a全部将>90%的VP 2完全切割成VP 3，但在空衣壳中不切割，并且不进一步消化衣壳。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将VP 2切割成VP 3，并且还在另外的内部位点切割，引起颗粒崩解和蛋白质降解。由蛋白酶K或枯草杆菌蛋白酶产生的几种部分消化产物类似于31- 32.5kDa，表明衣壳蛋白的环3内以及其他位点内的切割。蛋白酶处理的衣壳在pH 5.5或7.5没有显着改变其消化的敏感性。CPV空衣壳的等电点为pH 5.3，而完整衣壳的pH值比空衣壳的pH值高0.3，但在将VP 2蛋白水解为VP 3后，完整衣壳的pi与空衣壳的pi相同。针对各种衣壳蛋白序列的抗体显示，大多数VP 2分子的氨基末端在完整而非空颗粒的外部，VP 1独特序列在内部，并且衣壳可以通过热或尿素处理而解体以暴露内部序列。添加到细胞中的衣壳位于细胞质中的囊泡中，这些囊泡似乎是溶酶体。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。在用[S-35]甲硫氨酸标记的CPV衣壳在0 ℃下与细胞结合并使细胞升温后，即使在长时间孵育后也几乎没有观察到VPI或VP 2的裂解。在存在蛋白酶抑制剂的情况下用病毒接种细胞没有显著降低感染。(C)北京：科学出版社.
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 down arrow Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin a 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 similar to 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 [S-35]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VPI 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. (C) 1998 Academic Press.