Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly

Human immunodeficiency virus type 1 Gag protein is cotranslationally myrist oylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimeriz ation of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomeri zation process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multim eric nature of each Gag protein and the ability to form Gag virus-like part icles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of th e assembly competent Gag; (ii) the p2 domain, located at the capsid/nucleoc apsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag as sembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimeriz ation stages but plays a key role in conversion of these multimers to Gag V LP. We suggest that the Gag trimer and the >1,000-kDa multimer are intermed iates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VIE development a nd suggest that each stage involves a separate domain, MA, p2, or N-termina l myristoylation. each of which contributes to HIV particle assembly.