The bluetongue virus (BTV) minor protein VP4, with molecular mass of 76 kDa, is one of the seven structural proteins and is located within the inner capsid of the virion. The protein has a putative leucine zipper near the carboxy terminus of the protein. In this study, we have investigated the functional activity of this putative leucine zipper by a number of approaches. The putative leucine zipper region (amino acids [aa] 523 to 551) was expressed initially as a fusion protein by using the pMAL vector of Escherichia coli, which expresses a maltose binding monomeric protein. The expressed fusion protein was purified by affinity chromatography, and its size was determined by gel filtration chromatography. Proteins of two sizes, 51 and 110 kDa, were recovered, one equivalent to the monomeric form and the other equivalent to the dimeric form of the fusion protein. To prove that the VP4-derived sequence was responsible for dimerization of this protein, a mutated fusion protein was created in which a VP4 leucine residue (at aa 537) within the zipper was replaced by a proline residue. Analyses of the mutated protein demonstrated that the single mutation indeed prevented dimerisation of the protein. The dimeric nature of VP4 was further confirmed by using purified full-length BTV-10 VP4 recovered from recombinant baculovirus-expressing BTV-10 VP4-infected insect cells. Using chemical cross-linking and gel filtration chromatography, we documented that the native VP4 indeed exists as a dimer in solution. Subsequently, Leu537 was replaced by either a proline or an alanine residue and the full-length mutated VP4 was expressed in the baculovirus system. By sucrose density gradient centrifugation and gel filtration chromatography, these mutant forms of VP4 were shown to lack the ability to form dimers. The biological significance of the dimeric forms of VP4 was examined by using a functional assay system, in which the encapsidation activity of VP4 into core-like particles (CLPs) was studied (H. LeBlois, T. French, P. P. C. Mertens, J. N. Burroughs, and P. Roy, Virology 189:757-761, 1992). We demonstrated conclusively that dimerization of VP4 was essential for encapsidation by CLPs.