Figure 3.

The host-catalyzed HIV-1 assembly pathway was developed into a screen for small molecule inhibitors. (A) Schematic showing the host-catalyzed HIV-1 assembly pathway, starting with Gag synthesis and formation of the early ~10S assembly intermediate. The ~80S assembly intermediate is formed when ~10S Gag co-opts a host RNP complex containing ABCE1 and DDX6, two host enzymes that have been shown to facilitate assembly. The co-opted host RNP complex is distinct from but related to P-bodies and stress granules, which are larger host RNP complexes. The ~80S assembly intermediate targets to the plasma membrane where Gag multimerization continues, resulting in sequential formation of the ~150S and ~500S assembly intermediates and the fully assembled ~750S immature capsid. Upon completion of assembly, the host RNP complex is released. Relevant references are in the text. (B) A cell-free protein synthesis and assembly plate screen that recapitulates the host-catalyzed HIV-1 assembly pathway was developed and utilized to identify small molecule inhibitors of the pathway [29]. In this screen, a capture antibody directed against Gag binds Gag monomers, oligomers, and multimers generated in a cell-free assembly reaction. The same anti-Gag antibody is used as a detection antibody that binds to captured oligomers and multimers, but not monomers whose binding site is occupied. The signal produced by the detection antibody is proportional to the amount of anti-Gag binding with a larger fluorescent signal indicating more extensive multimerization. The upper diagram shows the large signal that is produced when the HIV-1 cell-free assembly reaction is carried out in the presence of DMSO, which does not inhibit Gag assembly. The lower diagram shows inhibition of that signal when an inhibitor of Gag assembly is added at the start of the cell-free reaction. Legend in the middle applies to panels (A) and (B).