In vitro assembly properties of wild-type and cyclophilin-binding defective human immunodeficiency virus capsid proteins in the presence and absence of cyclophilin A

The cellular protein cyclophilin A (CypA) binds specifically to the human i mmunodeficiency virus type 1 (HIV-1) capsid (CA) protein and is incorporate d into HIV-1 particles at a molar ratio of 1:10 (CypA/CA). Structural analy sis of a CA-CypA complex suggested that CypA may destabilize interactions i n the viral capsid and thus promote uncoating. We analyzed the influence of CypA on the in vitro assembly properties of wild-type (WT) CA and derivati ves containing substitutions of Gly89 in the Cyp-binding loop. All Variant proteins were significantly impaired in CypA binding. In the presence of Cy pA at a molar ratio of 1:10 (CypA/CA), WT CA assembled into hollow cylinder s that were similar to those observed in the absence of CypA but slightly l onger. Higher CypA concentrations inhibited cylinder formation. Variant CA proteins G89L and G89F yielded similar cylinders as the WT protein but were significantly more resistant to CypA. Cryoelectron microscopic analysis of WT cylinders assembled in the presence of CypA revealed direct binding of CypA to the outer surface. Electron diffraction patterns generated from the se cylinders indicated that CypA causes local disorder. The addition of Cyp A to preassembled cylinders had little effect, however, and cylinders were only disrupted when incubated with a threefold molar excess of CypA for sev eral hours. These results suggest that CypA does not efficiently destabiliz e CA interactions at the molar ratio observed in the virion and therefore i s unlikely to serve as an uncoating factor (C) 1999 Academic Press.