CHARACTERIZATION AND REPLICASE ACTIVITY OF DOUBLE-LAYERED AND SINGLE-LAYERED ROTAVIRUS-LIKE PARTICLES EXPRESSED FROM BACULOVIRUS RECOMBINANTS

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 determi ned by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and G TP binding assays, and their ability to support synthesis of negative- strand RNAs on positive-sense template RNAs was determined in an in vi tro replication system, Coexpression of all possible combinations of V P1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulte d in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-la yered VLPs, These VLPs had the structural characteristics of empty rot avirus double-layered particles and contained the indicated protein sp ecies, Only VP1/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VP1, VP2, and VP3, the p roteins 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 struc tural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs s upported 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 t he assembly of VP1 and VP3 and in replicase activity is most likely st ructural, (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.