BACULOVIRUS MULTIGENE EXPRESSION VECTORS AND THEIR USE FOR UNDERSTANDING THE ASSEMBLY PROCESS OF ARCHITECTURALLY COMPLEX VIRUS-PARTICLES

The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the larg e-scale production of recombinant proteins for a variety of applicatio ns including the structure-function analysis of genes and their gene p roducts. We have developed a number of baculovirus multigene expressio n vectors and utilized these to understand the assembly process of mul ticomponent capsid structures of large viruses such as bluetongue viru s (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 Angstrom in diameter and comprised of two protein she lls containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease t hat is sometimes fatal in certain parts of the world (e.g., Africa, As ia, and the Americas). Using baculovirus multigene vectors, we have co -expressed various combinations of BTV genes in insect cells and produ ced structures that mimic the various stages of BTV assembly. For exam ple, co-expressed VP3 and VP7 form BTV core-like particles, while co-e xpressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using de letion, point and domain switching analyses of each protein, we have b een able to identify certain sequences in the VP7 and VP3 proteins tha t are essential for the assembly of core-like particles. These express ion and biochemical studies have been complemented by collaboration st udies using cryoelectron microscopy and image processing analyses to p rovide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immun e responses elicited by these self-assembled particles, and chimeric d erivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 mu g of the self-assembled virus-like p articles, we have shown that they can confer long-lasting protection i n sheep against BTV.