Baculoviridae is a family of double-stranded DNA viruses which infect the larvae of insects such as butterflies and moths. Baculoviruses are widely used for the biological control of insect pests and as an expression system for eukaryotic recombinant proteins. The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the prototype baculovirus. Its genome is stored in an enveloped particle containing a rod-shaped nucleocapsid comprised of three parts, the cap, body, and base. Reconstruction of the AcMNPV nucleocapsid by cryo-EM shows that the nucleocapsid forms a helical tube of the major nucleocapsid protein (MCP, VP39) which forms a dimer with a novel fold and organization. A loop in VP39 forms a “lasso” that interlocks the two subunits in the dimer through an interaction with the VP39 “pin”, a projecting 2-strand beta-sheet. We hypothesized that two interactions are critical for assembly of VP39 into helical tubes: inter-dimer disulfide bonds and lasso-lasso interactions. We showed that mutation of the three cysteines forming inter-dimer disulfide bonds affects the ability of VP39 to self-assemble into virus-like particles. In addition, through mutagenesis of key residues involved in the lasso-lasso interaction, we confirmed that this interaction is required for assembly. Also, we determined the 1.6 Å-resolution crystal structure of the MCP through mutations for conserved three cysteine and key residues in inter-dimer interaction supporting our hypothesis structurally. These results provide a platform to understand the broader role of disulfide bond formation and the VP39 lasso in baculovirus assembly, stability and genome packaging.