Based on the essential role that the HIV core and its proper stability play in the regulation of reverse transcription in target cells, these observed core defects in NCINI progeny are likely to contribute significantly to the observed block in vDNA synthesis. T174I IN was among the resistance mutations selected by GS-A and GS-B and confers high-level resistance to NCINIs. Threonine 174 residue lines the NCINI-binding pocket within the IN dimer interface and makes significant contacts with the inhibitor molecule. Importantly, we show here that an HIV-1 variant with the T174I mutation was resistant to the latestage inhibitory effect of NCINIs. It is reasonable to conclude that VE-821 NCINIs exert the late-stage effect in newly produced virions via the same molecular interactions with the IN protein as those involved in mediating the substantially weaker inhibition of vDNA integration in target cells. The significant PF-4217903 difference in NCINI potency at the late vs early stage is, therefore, intriguing. Possibly, IN interactions with viral and/or host proteins co-packaged within the mature core restrict NCINI access to its binding pocket, either directly or via conformational changes that could reduce the binding affinity of NCNIs for the IN dimer interface. Indeed, a number of host proteins packaged in HIV particles have been shown to bind IN. Alternatively, the early-stage but not the late-stage effect of NCINIs may require their direct competition with LEDGF binding to IN, leading to the potency differential observed herein. Our observation that neither the overexpression nor the knockdown of LEDGF in the virus producer cells impacted virus infectivity or NCINI antiviral potency implies that the late-stage effect of these compounds does not involve direct competition with LEDGF binding to the IN domain during virus assembly. Although its essential role in vDNA integration has been firmly established, there is little evidence for the requirement of LEDGF during the production of infectious virus. A substantial knockdown of LEDGF in virus-producing cells was reported to minimally impact virus production or its infectivity, and proteomic studies did not identify LEDGF among host proteins packaged within HIV-1 particles. In addition, the progeny virus harboring a Q168A mutation in IN that disrupts its binding to LEDGF was replication defective due to a block in vDNA integration, but the mutant virions showed no late-stage defects and supported normal reverse transcription in target cells. Furthermore, although cell lines overexpressing the dominant negative LEDGF-derived IN binding domain were refractory to viral infection due to failed integration, progeny virus produced from the IBD overexpressing cells was reported to be fully infectious.