Mutations conferring HIV-1 resistance to these inhibitors map to the IN dimer interface within and near the LEDGF-binding pocket, confirming IN as the target for NCINIs. Importantly, these compounds were found to inhibit vDNA integration in target cells, albeit at high concentrations . In the present study, we used a series of virologic, biochemical, and imaging techniques to assess whether the interference with the vDNA integration is indeed the primary antiviral mode of action for NCINIs. Surprisingly, our results reveal that although the compounds still act through binding to the IN dimer interface, their antiviral effect is primarily derived during the late stage of virus replication via the promotion of IN multimerization and disruption of proper core maturation. These effects render the progeny virus noninfectious due to an inability to initiate vDNA synthesis following entry into the target cells. Our findings not only demonstrate a novel mode of action for the NCINI class, but also support the previously VE-822 proposed role of IN protein in the process of HIV assembly and maturation. To identify stages in the virus life cycle impacted by NCINIs, we utilized a two-part assay system that enabled the measurement of antiviral potency at the late vs early phase of the virus life cycle by altering the presence of compounds in cultures of virus producer and target cells. As expected, the integration inhibitor RAL was active only when present during infection of the target cells, whereas the late-acting protease inhibitor atazanavir exhibited antiviral activity only when present during virus production. Surprisingly, when the NCINIs GS-A and GS-B were restricted to only the target cell infection phase, both compounds showed a SCH772984 marked reduction in potency relative to that observed in the full-cycle assay. Importantly, and in striking contrast to RAL, restricting NCINI exposure to the virus-production stage of infection was sufficient to yield activity similar to that observed in the full-cycle assay. In addition, similar late-stage potencies were observed with virus produced from MT-2 T cell lines and closely matched activity observed in multi-cycle assays employing human primary PBMCs. The T174I mutation in IN confers resistance to the NCINI class of inhibitors. The T174I virus is substantially less sensitive to the late-stage inhibition of both GS-A and GS-B, but retains full sensitivity to RAL and ATV, confirming that the latestage antiviral effect of NCINIs is still driven by targeting the viral IN protein. We next investigated whether the late-stage effect of NCINIs involves any virucidal effect on virus progeny. We incubated cellfree mature virus with high concentrations of the inhibitor and then removed unbound compound by repeated dilution and ultrafiltration of the virus sample.