Incorporation of Pol into human immunodeficiency virus type 1 Gag virus-like particles occurs independently of the upstream Gag domain in Gag-Pol

By using particle-associated reverse transcriptase (RT) activity as an assay for Poll incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Poll missing cis Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20% of the level of wild-type Gag-Poll). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Poll that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70% of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Poll into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pot incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNA(Lys) into Gag VLPs, and primer tRNA(3)(Lys) is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Poll species still retains the capacity to bind to both Gag and tRNA(3)(Lys), which may be required for Poll and tRNA(3)(Lys) to be retained in the assembling virion until budding is completed.