Fig 1.

Primary MDM silencing assay. (A) Schematic of brain-derived macrophage-tropic HIV-1 reporter construct YU2/Δenv/nLuc. The destabilized nano-luciferase (nLuc) reporter was placed upstream of a P2A ribosomal skip site and inserted at the 5’ end of the nef gene of HIV-1 YU2. A premature stop was created in env by introduction of a 2-bp insertion at codon 59 of the open reading frame. (B) Overview of primary cell assay. Monocytes were cultured for 6 d to mature into MDM and then infected with the VSVg-pseudotyped reporter virus. They were cultured for 7 d more to allow infection to proceed, then re-plated into 384 well plates. One day later, test compounds were added and maintained in the cultures for 5 d. Cells were then harvested and nLuc for virus expression and CellGlo for cell viability measured in cell lysates of replicate plates. (C) Timing the completion of integration after reporter virus infection. The integrase inhibitor raltegravir (RTG) was added 1 d prior to, 1 d after, or 4 d after MDM were infected with the VSVg-pseudotyped reporter virus. By 4 d after infection, infection was resistant to integration blocking.