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Journal of Virology, October 2001, p. 9156-9164, Vol. 75, No. 19
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.19.9156-9164.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Proteolytic Processing of the P2/Nucleocapsid Cleavage Site Is Critical for Human Immunodeficiency Virus Type 1 RNA Dimer Maturation

M. Shehu-Xhilaga,1,2 H. G. Kraeusslich,3 S. Pettit,4 R. Swanstrom,5 J. Y. Lee,6 J. A. Marshall,6 S. M. Crowe,1,2 and J. Mak1,7,*

AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria,1 Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria,6 and Department of Biochemistry and Molecular Biology7 and Department of Medicine,2 Monash University, Clayton, Victoria, Australia; Abteilung Virologie, Universität Heidelberg, Heidelberg, Germany3; and Department of Biochemistry and Biophysics5 and Department of Medicine, Infectious Diseases,4 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Received 22 January 2001/Accepted 25 June 2001

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

* 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, October 2001, p. 9156-9164, Vol. 75, No. 19
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.19.9156-9164.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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