Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

被引:78
作者
Zeng, CQY
Wentz, MJ
Cohen, J
Estes, MK
Ramig, RF
机构
[1] BAYLOR COLL MED,DIV MOLEC VIROL,HOUSTON,TX 77030
[2] INRA,CTR RECH JOUY EN JOSAS,LAB VIROL & IMMUNOL MOL,F-78350 JOUY EN JOSAS,FRANCE
关键词
D O I
10.1128/JVI.70.5.2736-2742.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Rotavirus has a capsid composed of three concentric protein layers, We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system, Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs, These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species, Only VP1/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VP1, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VPI is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.
引用
收藏
页码:2736 / 2742
页数:7
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