FIG 1.

HIV-1 life cycle, assembly pathway, and assembly plate screen. (A) Schematic showing each step of the HIV-1 life cycle, beginning with expression of the integrated provirus, followed by the late and early stages of the life cycle, ending with integration in a newly infected cell. The different stages of the virus life cycle are indicated in blue text. Examples of ARV drugs currently in use are in red text, with black labels under blockade arrows indicating the targets of these drugs. The pink line indicates late events in the viral life cycle that are not targeted by currently approved ARV drugs. (B) Schematic showing the host-catalyzed HIV-1 assembly pathway, starting with Gag synthesis and formation of the early ∼10S assembly intermediate. Next, the cytosolic ∼80S intermediate forms when ∼10S Gag coopts a host RNP complex that contains ABCE1 and the RNA granule protein DDX6, two host enzymes that have been shown to facilitate assembly. The ∼80S assembly intermediate appears to target to the plasma membrane where Gag multimerization continues resulting in formation of the ∼150S and subsequently the ∼500S late assembly intermediate. When assembly of ∼750S immature capsid is completed, the host RNP complex is released. Relevant references are in the text. (C) Schematic showing the cell-free protein synthesis and assembly plate screen that was utilized to identify small molecule inhibitors of the host-catalyzed pathway of HIV-1 assembly. Briefly, anti-Gag antibody (capture antibody) binds Gag monomers, oligomers, and multimers generated in a cell-free assembly reaction. The same Gag antibody is used as a detection antibody that binds to captured oligomers and multimers, but not monomers, in proportion to the amount of multimerization, thereby generating a larger fluorescent signal when multimerization occurs. The upper diagram shows anti-Gag antibodies capturing and detecting Gag oligomers and multimers in assembly intermediates formed during an HIV-1 cell-free assembly reaction carried out in the presence of DMSO, which does not inhibit assembly. The lower diagram shows that adding an assembly inhibitor at the start of the cell-free reaction causes fewer Gag oligomers and multimers to be produced, thereby reducing the detection antibody signal relative to signal in the DMSO control.