Identification and characterization of virus assembly intermediate complexes in HIV-1-infected CD4+ T cells

For type-C and lentiviruses, including human immunodeficiency virus type 1 (HIV-1), the pathway of virus assembly remains poorly defined, and the assembly and budding of capsids are believed to occur simultaneously at the plasma membrane of the infected cell. We have now identified two putative HIV-1 assembly intermediate complexes in infected CD4+ T cells. The first of these intermediates, a detergent-resistant complex (DRC), was identified as a large oligomer that had a density of 1.10-1.13 g/ml and was primarily composed of Pr55Gag and Pr160Gag-Pol precursors. The other putative intermediate was a detergent-sensitive complex (DSC) with a density of 1.15-1.17 g/ml, which apparently represented the products of extensive proteolytic processing of both the Pr55Gag and Pr160Gag-Pol precursors. Both complexes could be distinguished from released mature virions as well as immature viral particles. Surprisingly, the formation of DRC was not dependent upon the myristylation at the N-terminus of the Gag proteins, a signal required for plasma membrane targeting and virus production. However, the myristic acid modification was essential for the formation of DSC. These data suggest that interactions between individual Gag molecules and between Gag and Gag-Pol precursors may occur before their targeting to the plasma membrane during HIV-1 assembly. However, formation of the late virus assembly complex and productive processing of Pr55Gag and Pr160Gag-Pol precursors apparently do not occur until these precursors are targeted to the plasma membrane.