Methods for the study of HIV-1 assembly.

Virus assembly constitutes a key phase of the HIV-1 replication cycle. The assembly process is initiated by the synthesis of the Gag precursor protein, Pr55(Gag), in the cytosol of the infected cell. After its synthesis, Pr55(Gag) is rapidly transported in most cell types to the plasma membrane (PM) where it associates with the inner leaflet of the lipid bilayer. Gag-Gag interactions lead to the assembly of an electron-dense patch of Gag proteins at the membrane. The viral envelope (Env) glycoproteins associate with Gag during the assembly process. The highly multimerized Gag complex begins to bud outwardly from the PM and eventually pinches off from the cell surface. Concomitant with release, the viral protease cleaves Pr55(Gag) to the mature Gag proteins matrix, capsid, nucleocapsid and p6, leading to core condensation. The mature infectious virus particle is now able to initiate a new round of infection in a fresh target cell. Techniques have been developed in many laboratories to study each of the distinct phases of the HIV-1 assembly and release pathway. A number of these techniques are described in detail in this chapter.