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Journal of Virology, November 2002, p. 10598-10607, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10598-10607.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Nuclear Localization of Human Immunodeficiency Virus Type 1 Preintegration Complexes (PICs): V165A and R166A Are Pleiotropic Integrase Mutants Primarily Defective for Integration, Not PIC Nuclear Import

Ana Limón,1,2 Eric Devroe,3,4 Richard Lu,1,2 Hina Z. Ghory,1,2 Pamela A. Silver,3,4 and Alan Engelman1,2*

Departments of Pathology,1 Biological Chemistry and Molecular Pharmacology, Harvard Medical School,3 Departments of Cancer Immunology and AIDS,2 Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 021154

Received 21 May 2002/ Accepted 15 July 2002

Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome. A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells. One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types. In this study we confirmed that HIV-1V165A and HIV-1R166A were replication defective and that less mutant viral cDNA localized to infected cell nuclei. However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import. First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei. Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1V165A and HIV-1R166A, suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses. Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli. Based on these results, we conclude that HIV-1V165A and HIV-1R166A are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells.

* Corresponding author. Mailing address: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Phone: (617) 632-4361. Fax: (617) 632-3113. E-mail: alan_engelman{at}dfci.harvard.edu.

Journal of Virology, November 2002, p. 10598-10607, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10598-10607.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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