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-1. However, the mechanisms underlying its role in virus particle production are still unclear. Recently, it was reported that Pr55Gag localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X100 (TX100). In this study, we sought to identify the relationship between CA-NC spacer-region mediated Gag assembly and membrane targeting.

Methods: Membrane binding efficiency is assessed by membrane floatation assays and confocal microscopy. To study the newly synthesized HIV-1 Gag on the plasma membrane, we combined pulse-chase techniques with sucrose density gradient centrifugation assays. The plasma membrane characteristics are determined by treating Gag associated membranes with nonionic detergent, i.e. Brij98. Assembly of wild-type and mutant Gag proteins are studied by in-vitro assembly system.

Results: Mutation of the CA-NC spacer led to dramatic reductions in virus production, which was mainly attributed to severely attenuated association of the mutated Gag protein with the plasma membrane. Analysis of newly synthesized HIV Gag showed that mutations of the CA-NC spacer resulted in different distribution pattern on the plasma membrane: Pr55Gag are mainly detected in the 40% and the top of the 50% fractions, whereas the mutated Gag, i.e. M368A, had more broad distribution patterns. Further exposure of membrane-associated Gag protein to Brij98 revealed that M368A Gag was mainly localized on Brij98 sensitive membranes containing small complexes, whereas the wild-type Pr55Gag was mainly associated with Brij98 resistant membrane within large complexes. Interestingly, the defects of membrane targeting associated with the M368A mutation can be corrected by co-transfection with a plasmid expressing wild-type Gag. In addition, in-vitro assembly assays showed that the M368A mutant had dramatically decreased ability to form particles compared with wild-type Gag.

Conclusions:  Our data further demonstrate that the CA-NC spacer region mediates Gag assembly and this feature is required for efficient targeting of Gag to the plasma membrane. Based on the essential and special role of CA-NC spacer in HIV-1 life cycle, it may represent an attractive target for the development of novel therapeutic strategies.

Keywords: HIV Assembly; Membrane Targeting; Lipid Raft