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Journal of Virology, February 2001, p. 1834-1841, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1834-1841.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Maintenance of the Gag/Gag-Pol Ratio Is Important for Human Immunodeficiency Virus Type 1 RNA Dimerization and Viral Infectivity

Miranda Shehu-Xhilaga,1,2 Suzanne M. Crowe,1,2,3 and Johnson Mak1,4,*

AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield,1 Department of Biochemistry and Molecular Biology4 and Department of Medicine,2 Monash University, Clayton, and National Centre for HIV Virology Research, Melbourne,3 Victoria, Australia

Received 11 August 2000/Accepted 21 November 2000

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.

* Corresponding author. Mailing address: AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria, Australia 3078. Phone: 61 3 9282 2217. Fax: 61 3 9482 6152. E-mail: mak{at}burnet.edu.au.

Journal of Virology, February 2001, p. 1834-1841, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1834-1841.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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