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

doi:10.1128/JVI.75.4.1834-1841.2001

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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 frameshiftingevent. In infected cells, this generates Gag and Gag-Pol in aratio that is estimated to be 20:1, a ratio that is conservedamong retroviruses. To examine the impact of this ratio on HIV-1replication and viral assembly, we altered the Gag/Gag-Pol ratioin virus-producing cells by cotransfecting HIV-1 proviral DNAwith an HIV-1 Gag-Pol expression vector. Two versions of the Gag-Polexpression vector were used; one contains an active protease [PR(+)],and the other contains an inactive protease [PR( $-$ )]. In an attemptto produce viral particles with Gag/Gag-Pol ratios ranging from20:21 to 20:1 (wild type), 293T cells were cotransfected withvarious ratios of wild-type proviral DNA and proviral DNA fromeither Gag-Pol expression vector. Viral particles derived fromcells with altered Gag/Gag-Pol ratios via overexpression of PR( $-$ )Gag-Pol showed a ratio-dependent defect in their virion proteinprofiles. However, the defects in virion infectivity were independentof the nature of the Gag-Pol expression vector, i.e., PR(+) orPR( $-$ ). Based on equivalent input of reverse transcriptase activity,we estimated that HIV-1 infectivity was reduced 250- to 1,000-foldwhen the Gag/Gag-Pol ratio in the virion-producing cells was alteredfrom 20:1 to 20:21. Although virion RNA packaging was not affectedby altering Gag/Gag-Pol ratios, changing the ratio from 20:1 to20:21 progressively reduced virion RNA dimer stability. The impactof the Gag/Gag-Pol ratio on virion RNA dimerization was amplifiedwhen the Gag-Pol PR( $-$ ) expression vector was expressed in virion-producingcells. Virions produced from cells expressing Gag and Gag-PolPR( $-$ ) in a 20:21 ratio contained mainly monomeric RNA. Our observationsprovide the first direct evidence that, in addition to proteolyticprocessing, the ratio of Gag/Gag-Pol proteins is also importantfor RNA dimerization and that stable RNA dimers are not requiredfor 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 94826152. 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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