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 precursor protein results from a -1 ribosomal frameshifting event. In infected cells, 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 this ratio on HIV-1 replication and viral assembly, we altered the Gag/Gag-Pol ratio in virus-producing cells by cotransfecting HIV-1 proviral DNA with an HIV-1 Gag-Pol expression vector. Two versions of the Gag-Pol expression vector were used; one contains an active protease [PR(+)], and the other contains an inactive protease [PR(-)]. In an attempt to produce viral particles with Gag/Gag-Pol ratios ranging from 20:21 to 20:1 (wild type), 293T cells were cotransfected with various ratios of wild-type proviral DNA and proviral 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. However, 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-producing 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 the Gag/Gag-Pol ratio on virion RNA dimerization was amplified when the Gag-Pol PR(-) expression vector was expressed in virion-producing cells. Virions produced from cells expressing Gag and Gag-Pol PR(-) in a 20:21 ratio contained mainly monomeric RNA. Our observations provide the first direct evidence that, in addition to proteolytic processing, the ratio of Gag/Gag-Pol proteins is also important for RNA dimerization and that stable RNA dimers are not required for encapsidation of genomic RNA in HIV-1.
Shehu-Xhilaga
M
M
AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria, Australia.
Crowe
S M
SM
Mak
J
J
D002460:Cell Line; D019281:Dimerization; D015685:Fusion Proteins, gag-pol / Q000378:metabolism*; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D012367:RNA, Viral; D014764:Viral Proteins