Human immunodeficiency virus-type 1 (HIV-1) remains one of the leading contributors to the global burden of disease, and novel antiretroviral agents with alternative mechanisms are needed to cure this infection. Here, we describe an exploratory attempt to optimize the antiretroviral properties of benfluron, a cytostatic agent previously reported to exhibit strong anti-HIV activity likely based on inhibitory actions on virus transcription and Rev-mediated viral RNA export. After obtaining six analogs designed to modify the benzo[c]fluorenone system of the parent molecule, we examined their antiretroviral and toxicity properties together with their capacity to recognize the Rev Recognition Element (RRE) of the virus RNA and inhibit the RRE–Rev interaction. The results indicated that both the benzo[c] and cyclopentanone components of benfluron are required for strong RRE–Rev target engagement and antiretroviral activity and revealed the relative impact of these moieties on RRE affinity, RRE–Rev inhibition, antiviral action and cellular toxicity. These data provide insights into the biological properties of the benzo[c]fluorenone scaffold and contribute to facilitating the design of new anti-HIV agents based on the inhibition of Rev function.

The complex between several monomers of the HIV-1 Rev protein and the Rev Response Element (RRE) in the viral RNA allows nuclear export of unspliced or singly-spliced viral transcripts, an essential step in the virus cycle that is not targeted by any of the currently marketed antiretroviral treatments. The RRE adopts a multi-domain structure whose three-dimensional details are currently unknown at atomic resolution. In order to shed light on RRE structure and on the mechanism of RRE-Rev complex assembly, we set up a method based on fluorescence resonance energy transfer (FRET) that has allowed us to measure RRE interdomain distance as a function of solution conditions and changes in helix length and junction nucleotides. By combining this information with all-atom ensemble modeling, comparative sequence analyses and electrophoretic band-shift assays assessing Rev association, we clarify the roles of helices and junctions on the spatial organization of RRE domains and RRE-Rev complex formation and propose an all-atom model of RRE structure consistent with the available evidence.