The virus-associated human immunodeficiency virus type 1 Gag-Pol carrying an active protease domain in the matrix region is severely defective both in autoprocessing and in trans processing of gag particles.

We have previously demonstrated that a human immunodeficiency virus (HIV) chimeric Gag protein containing a partial replacement of the matrix domain by the viral protease domain (PR) could undergo autoprocessing with no virus particle production [J. Virol. 74 (2000) 3418]. To further analyze the effects of repositioned PR on virus particle production and Gag-Pol incorporation, we introduced the chimeric PR construct into a PR-negative Gag-Pol expression plasmid and coexpressed the resultant construct with a Pr55(gag) expression plasmid (pGAG) in 293T cells. Analysis indicated that the chimeric PR was similar to native PR in that both could prevent virus particle production in cotransfections with an equivalent amount of pGAG plasmid DNA, suggesting an efficient trans processing of Pr55(gag) by the chimeric PR. In cotransfections with the pGAG at a DNA ratio of 1:10 to 1:20, which resembles the normal intracellular expression ratio of Gag-Pol to Gag, Gag-Pol carrying the PR in the Gag coding region could undergo autoprocessing in cells and was incorporated into virus particles at a level about 20-40% of that of wild-type Gag-Pol. However, the incorporated chimeric Gag-Pol was unable to autocleave and unable to process the Gag particles properly, as mature particle-associated reverse transcriptase (RT) and p24(gag) proteins were barely detected. Our data strongly suggest that positioning an active HIV PR in the matrix region significantly affects the PR-mediated virus particle maturation.