Rotaviruses are triple-layered particles that contain four major capsid proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP3. We have cloned each of the rotavirus genes coding for a major capsid protein into the baculovirus expression system and expressed each protein in insect cells. Coexpression of different combinations of the rotavirus 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 were similar to double-layered rotavirus particles. Coexpression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of nonneutralizing and neutralizing 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 interacts with VP6. Cell binding assays performed with each of the VLPs indicated that VP4 is the viral attachment protein. Chimeric particles containing VP7 from two different G serotypes also were obtained. The ability to express individual proteins or to coexpress different subsets 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.

The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis.

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The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for doublestranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified singleshelled particles.

C57BL/6 (1-1-2 b) mice were primed with the bovine RF strain of rotavirus to study the induction of CD8 + cytotoxic T lymphocytes (CTLs). These rotavirusspecific CTLs were detected only after in vitro restimulation with the virus. Using a recombinant vaccinia virus we identified the RF VP7 protein as a major target of these CTLs. The response against this protein was obtained also after in vitro restimulation with simian SA11 and human WA strains of rotavirus. Using published D b and K b allele-specific motifs to predict possible CTL epitopes in the RF VP7 protein, we synthesized and tested 18 predicted peptides of VP7. Only one peptide was able to sensitize target cells at a concentration below 5 × 10 -7 M. This CTL epitope was also induced by immunization with the RF VP7 expressed with a baculovirus vector, and was shown to be immunodominant by its capacity to inhibit, in an unlabelled target assay, the bulk response against ceils infected with recombinant vaccinia virus expressing VP7. This CTL epitope overlaps the H2 signal peptide of the protein.

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