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Journal of Virology, September 2007, p. 10047-10054, Vol. 81, No. 18
0022-538X/07/$08.00+0     doi:10.1128/JVI.02496-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1 ^,

Lori V. Coren, James A. Thomas, Elena Chertova, Raymond C. Sowder II, Tracy D. Gagliardi, Robert J. Gorelick, and David E. Ott^*

AIDS Vaccine Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-1201

Received 13 November 2006/ Accepted 7 July 2007

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6^Gag proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9^NC/SP2 protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8^SP2/p6 or p15^NC/SP2/p6 protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6^Gag site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6^Gag and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9^NC/SP2 species in some aspect of integration.

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* Corresponding author. Mailing address: AIDS Vaccine Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201. Phone: (301) 846-5723. Fax: (301) 846-5588. E-mail: ott{at}ncifcrf.gov

Published ahead of print on 18 July 2007.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, September 2007, p. 10047-10054, Vol. 81, No. 18
0022-538X/07/$08.00+0     doi:10.1128/JVI.02496-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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