AEGiS-15IAC: Aberrant membrane binding and impaired multimerization of human immunodeficiency virus type 1 Gag caused by CA-NC spacer region mutations.

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Aberrant membrane binding and impaired multimerization of human immunodeficiency virus type 1 Gag caused by CA-NC spacer region mutations.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. TuOrA1135)

Guo X, Roldan A, Hu J, Wainberg MA, Liang C
McGill AIDS Centre/McGill University, Montreal, Canada

BACKGROUND: Lentiviral Gag protein contains a short spacer sequence that separates the capsid (CA) from the downstream nucleocapsid (NC) domain, which has been shown to play an important role in the assembly of HIV-1gag protein. However, the underlying mechanisms remain unclear. The objective of this study is to gain further insight into the mechanism underlying the effects that this CA-NC spacer region exerts during HIV-1 Gag membrane binding and assembly.

METHODS: Binding of HIV-1 Gag to the cellular membrane was assessed by membrane flotation assays as well as confocal microscopy. To study the newly synthesized HIV-1 Gag on the plasma membrane, we pulse-labeled Gag proteins with [s35] Met, which were subsequently fractionated in a sucrose flotation gradient. Multimerization of wild type or mutant Gag proteins were studied either within cells or using purified recombinant proteins.

RESULTS: The results of membrane flotation experiments showed that the SP1 mutations led to severely decreased levels of membrane-bound Gag proteins. In addition, the residual amounts of mutant Gag-membrane complexes were of lower density and sensitive to Brij 98 extraction in contrast to the Brij 98 -resistant membrane-bound wild-type Gag. This indicates that the membrane-associated SP1-mutated Gag proteins have been mostly retained on the non-raft microdomain. In the mean time, the same SP1 mutations dramatically inhibited Gag multimerization as shown by the results of experiments that were performed to characterize Gag assembly either within cells or using purified recombinant Gag proteins. Presumably, this assembly deficit may have, at least partially accounted for the diminished membrane binding associated with the SP1 mutations.

CONCLUSIONS: Our data further demonstrate that the CA-NC spacer region mediates Gag assembly and this feature is required for efficient binding of Gag to the plasma membrane. Due to its novel function during HIV-1 Gag assembly, the CA-NC spacer region may represent an attractive target for the development of novel therapeutic strategies.

Keywords: AEGIS, Genes, gag, Gene Products, gag, HIV-1, Mutation, Capsid, Membranes, Nucleocapsid, Virion, Cell Membrane, Nucleocapsid Proteins, Membrane Proteins, Humans, genetics, pharmacokinetics, metabolism

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