CHARACTERIZATION OF VIRUS-LIKE PARTICLES PRODUCED BY THE EXPRESSION OF ROTAVIRUS CAPSID PROTEINS IN INSECT CELLS

Rotaviruses are triple-layered particles that contain four major capsi d proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP 3. We have cloned each of the rotavirus genes coding for a major capsi d protein into the baculovirus expression system and expressed each pr otein in insect cells. Coexpression of different combinations of the r otavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The coexpression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which w ere similar to double-layered rotavirus particles. Coexpression of VP2 , VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 o r VP2/4/6/7 VLPs, which were similar to native infectious rotavirus pa rticles. The VLPs maintained the structural and functional characteris tics of native particles, as determined by electron microscopic examin ation of the particles, the presence of nonneutralizing and neutralizi ng epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4 /6/7 VLPs. The production of VP2/4/6 particles indicated that VP4 inte racts with VP6. Cell binding assays performed with each of the VLPs in dicated that VP4 is the viral attachment protein. Chimeric particles c ontaining VP7 from two different G serotypes also were obtained. The a bility to express individual proteins or to coexpress different subset s of proteins provides a system with which to examine the interactions of the rotavirus structural proteins, the role of individual proteins in virus morphogenesis, and the feasibility of a subunit vaccine.