Background:  The Gag protein of HIV-1 contains a 14-amino acid region, termed SP1, between the capsid and nucleocapsid domains. The importance of this short region is demonstrated by the evidence that either a M368A substitution within SP1 or the ΔSP1 deletion impaired virus production. The objective of this study is to determine at which stage the M368A mutation and the ΔSP1 deletion restrict Gag assembly.

Methods:  COS-7 cells were transfected using Lipofectamine. Binding of HIV-1 Gag to the cellular membranes was assessed by membrane flotation assays. Samples were fractionated on SDS-10% polyacrylamide gels and assessed by Western blots using mouse anti-p24 antibodies.

Results:  The M368A mutation, but not ΔSP1, severely diminished the levels of Gag-membrane association. This membrane-binding defect resulted from M368A was corrected either by a L364A second-site mutation or by changing nucleocapsid to the leucine zipper motif (LZ) derived from the yeast transcription factor GCN4. Yet, the resultant L364A-M368A and M368A-LZ Gag proteins remained defective in virus production with L364A-M368A exhibiting a more severe defect. Taken together, the M368A point mutation and the ΔSP1 deletion impair HIV-1 production by affecting different stages of Gag assembly. M368A restricts Gag-membrane association whereas deletion of SP1 affects a late step post Gag-membrane binding such as capsid morphogenesis or virus budding.

Conclusions:  Our data indicate that the SP1 region plays 2 distinct roles in HIV-1 assembly: targeting Gag to cellular membrane and promoting Gag multimerization on the membrane. Considering the vital role of SP1 in HIV-1 replication, this short motif may represent a potential target for HIV-1 intervention.  

Keywords: HIV-1; SP1; Assembly