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Journal of Virology, June 2005, p. 7756-7767, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7756-7767.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Single Point Mutations in the Zinc Finger Motifs of the Human Immunodeficiency Virus Type 1 Nucleocapsid Alter RNA Binding Specificities of the Gag Protein and Enhance Packaging and Infectivity

Michal Mark-Danieli,¹ Nihay Laham,¹ Michal Kenan-Eichler,¹ Asher Castiel,¹ Daniel Melamed,¹ Meytal Landau,² Nicole M. Bouvier,^3, Matthew J. Evans,^3,4, and Eran Bacharach^1*

Department of Cell Research and Immunology,¹ Department of Biochemistry, Tel Aviv University, Tel Aviv 69778, Israel,² Department of Biochemistry and Molecular Biophysics,³ Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University, New York, New York 10032⁴

Received 6 December 2004/ Accepted 15 February 2005

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal () is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.

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* Corresponding author. Mailing address: Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-640-7692. Fax: 972-3-642-2046. E-mail: eranbac{at}post.tau.ac.il.

Present address: Department of Internal Medicine, Mount Sinai School of Medicine, New York, NY 10029.

Present address: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10021.

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Journal of Virology, June 2005, p. 7756-7767, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7756-7767.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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