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Journal of Virology, December 2002, p. 11853-11865, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.11853-11865.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNA Incorporation Is Critical for Retroviral Particle Integrity after Cell Membrane Assembly of Gag Complexes

Shainn-Wei Wang and Anna Aldovini*

Department of Medicine, Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115

Received 17 April 2002/ Accepted 22 August 2002

The nucleocapsid (NC) domain of retroviruses plays a critical role in specific viral RNA packaging and virus assembly. RNA is thought to facilitate viral particle assembly, but the results described here with NC mutants indicate that it also plays a critical role in particle integrity. We investigated the assembly and integrity of particles produced by the human immunodeficiency virus type 1 M1-2/BR mutant virus, in which 10 of the 13 positive residues of NC have been replaced with alanines and incorporation of viral genomic RNA is virtually abolished. We found that the mutations in the basic residues of NC did not disrupt Gag assembly at the cell membrane. The mutant Gag protein can assemble efficiently at the cell membrane, and viral proteins are detected outside the cell as efficiently as they are for the wild type. However, only ~10% of the Gag molecules present in the supernatant of this mutant sediment at the correct density for a retroviral particle. The reduction of positive charge in the NC basic domain of the M1-2/BR virus adversely affects both the specific and nonspecific RNA binding properties of NC, and thus the assembled Gag polyprotein does not bind significant amounts of viral or cellular RNA. We found a direct correlation between the percentage of Gag associated with sedimented particles and the amount of incorporated RNA. We conclude that RNA binding by Gag, whether the RNA is viral or not, is critical to retroviral particle integrity after cell membrane assembly and is less important for Gag-Gag interactions during particle assembly and release.

* Corresponding author. Mailing address: Department of Medicine, Children's Hospital, and Department of Pediatrics, Harvard Medical School, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 355-8426. Fax: (617) 566-4721. E-mail: anna.aldovini{at}tch.harvard.edu.

Journal of Virology, December 2002, p. 11853-11865, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.11853-11865.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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