Background: The CA domain of the HIV-1 Gag polyprotein plays vital roles in both early and late phases of the viral life cycle and is therefore an attractive and novel antiviral target. Recently identified were 2 compounds (CAP-1 and CAP-2) with antiviral activity that bind to the N-terminal domain of CA (CA^N) and inhibit capsid assembly during viral maturation. Understanding how the CAP compounds bind to CA^N will help reveal their modes of action.

Methods: X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy were used in combination to elucidate the structure.

Results: We have determined the structure of the complex between CA^N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N′-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The global CA^N protein structure is unchanged, but undergoes a striking localized conformational change upon CAP-1 binding, in which the aromatic ring of Phe32 is displaced from its buried position in the protein core, thereby opening a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure

Conclusions: Small molecules of the CAP family can bind in a hydrophobic pocket located between CA^N helices 1, 2, 4, and 7 that is not observed in the native CA^N structure. CAP binding blocks capsid assembly, maturation, and viral infectivity, apparently by disrupting the formation of an essential interface between the CA^N and CA^C domains. Our work therefore reveals the presence of a previously unrecognized inhibitor binding pocket and can help guide optimization of inhibitors that can bind in this site.