Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity

Production of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol precu rsor protein results from a -1 ribosomal frameshifting event. In infected c ells, this generates Gag and Gag-Pol in a ratio that is estimated to be 20: 1, a ratio that is conserved among retroviruses. To examine the impact of t his ratio on HTV-1 replication and viral assembly, we altered the Gag/Gag-P ol ratio in virus-producing cells by cotransfecting HIV-1 proviral DNA with an HTV-1 Gag-Pol expression vector. Two versions of the Gag-Pol expression vector were used; one contains an active protease [PR(+)], and the other c ontains an inactive protease [PR(-)]. In an attempt to produce viral partic les with Gag/Gag-Pol ratios ranging: from 20:21 to 20:1 (wild type), 293T c ells were cotransfected with various ratios of wild-type proviral DNA and p roviral DNA from either Gag-Pol expression vector. Viral particles derived from cells with altered Gag/Gag-Pol ratios via overexpression of PR(-) Gag- Pol showed a ratio-dependent defect in their virion protein profiles. Howev er, the defects in virion infectivity were independent of the nature of the Gag-Pol expression vector, i.e., PR(+) or PR(-), Based on equivalent input of reverse transcriptase activity, we estimated that HIV-1 infectivity was reduced 250- to 1,000-fold when the Gag/Gag-Pol ratio in the virion-produc ing cells was altered from 20:1 to 20:21. Although virion RNA packaging was not affected by altering Gag/Gag-Pol ratios, changing the ratio from 20:1 to 20:21 progressively reduced virion RNA dimer stability. The impact of th e Gag/Gag-Pol ratio on virion RNA dimerization was amplified when the Gag-P ol PR(-) expression vector was expressed in virion-producing cells. Virions produced from cells expressing Gag and Gag-Pol PR(-) in a 20:21 ratio cont ained mainly monomeric RNA. Our observations provide the first direct evide nce that, in addition to proteolytic processing, the ratio of Gag/Gag-Pol p roteins is also important for RNA dimerization and that stable RNA dimers a re not required for encapsidation of genomic RNA in HIV-1.