STUDIES ON THE STRUCTURE AND FUNCTION OF NONSTRUCTURAL PROTEINS OF ROTAVIRUS

1.从表达正常NSP1的A5-13、编码40个氨基酸的NSP1的A5-10和缺失500个碱基的NSP1基因的A5-16中分离到产生大、中、小斑块的亚克隆。所有克隆均在MA-104细胞中复制良好，并能引起乳鼠腹泻。2.首次测定了人轮状病毒KU株基因组的全序列。3.在杆状病毒表达系统中表达了轮状病毒的6个结构蛋白VP1-VP4、VP6、VP7和5个非结构蛋白中的4个。建立了His标记的非结构蛋白的纯化程序，为精确检测非结构蛋白的功能提供了可能。4.通过VP2和VP6或VP2、VP6和VP7的共表达，获得了自组装的、人工的、空的单壳或双壳颗粒。此外，VP1、VP3、VP6、VP7、VP4以及VP2的共表达导致了缺乏基因组的人工病毒样颗粒的形成。5.在乳鼠口服感染实验中，观察了人轮状病毒G1、G3、G4、G8和G12株小鼠的腹泻情况。这一发现表明，利用人轮状病毒在小鼠身上进行轮状病毒感染保护实验是可能的。

1.We isolaed subclones producing large, medium, or small plaques from each of A5-13 expressing normal NSP1, A5-10 encoding omly 40 amino acid-NSP1, and A5-16 having NSP1 gene with 500 bp deletion. All of the clones replicated well in MA-104 cells, and induced dirrhea in suckling mice by oral administration. These results indicate that the insertion of foreign gene into NSP1 gene will be possible when the system of artificial infectious virus particles is established.2.We first determined the complete nucleotide sequence of the genome of human ratavirus (strain KU).3.All of the 6 structural proteins VP1-VP4, VP6, VP7 and 4 of 5 non-structural proteins of human rotavirus were expressed in baculovirus expression system. The produre for purification of his-tagged nonstructural proteins was established, and this enabled the precise examination of the function of nonstructural proteins.4.Self-assembled, artificial and empty single-shelled particles or double-shelled particules were obtained by coexpressions of VP2 and VP6, or VP2, VP6 and VP7. In addition, the coexpression of VP1, VP3, VP6, VP7, and VP4 as well as VP2 resulted in the formation of artificial virus-like particles lacking genome. These artificial empty particles have the value as a value as a vaccine candidate and a tool for studying the immunity of rotavirus infection.5.In the infection experiments by oral inoculation to suckling mice, we observed the diarrhea in the mice administered with G1, G3, G4, G8, and G12 human rotavirus strains. This finding show that it is possible to carry out the rotavirus infection-protection experiments in mice by using human rotaviruses.