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Journal of Virology, December 2004, p. 12735-12746, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.12735-12746.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Class II Integrase Mutants with Changes in Putative Nuclear Localization Signals Are Primarily Blocked at a Postnuclear Entry Step of Human Immunodeficiency Virus Type 1 Replication{dagger}

Richard Lu,1 Ana Limón,1,2,{ddagger} Eric Devroe,3,4 Pamela A. Silver,3,4 Peter Cherepanov,1,2 and Alan Engelman1,2*

Departments of Cancer Immunology and AIDS,1 Cancer Biology, Dana-Farber Cancer Institute,3 Departments of Pathology,2 Systems Biology, Harvard Medical School, Boston, Massachusetts4

Received 20 May 2004/ Accepted 6 August 2004

Integrase has been implicated in human immunodeficiency virus type 1 (HIV-1) nuclear import. Integrase analyses, however, can be complicated by the pleiotropic nature of mutations: whereas class I mutants are integration defective, class II mutants display additional assembly and/or reverse transcription defects. We previously determined that HIV-1V165A, originally reported as defective for nuclear import, was a class II mutant. Here we analyzed mutants containing changes in other putative nuclear localization signals, including 186KRK188/211KELQKQITK219 and Cys-130. Previous work established HIV-1K186Q, HIV-1Q214L/Q216L, and HIV-1C130G as replication defective, but phenotypic classification was unclear and nuclear import in nondividing cells was not addressed. Consistent with previous reports, most of the bipartite mutants studied here were replication defective. These mutants as well as HIV-1V165A synthesized reduced cDNA levels, but a normal fraction of mutant cDNA localized to dividing and nondividing cell nuclei. Somewhat surprisingly, recombinant class II mutant proteins were catalytically active, and class II Vpr-integrase fusion proteins efficiently complemented class I mutant virus. Since a class I Vpr-integrase mutant efficiently complemented class II mutant viruses under conditions in which class II Vpr-integrases failed to function, we conclude that classes I and II define two distinct complementation groups and suggest that class II mutants are primarily defective at a postnuclear entry step of HIV-1 replication. HIV-1C130G was also defective for reverse transcription, but Vpr-integraseC130G did not efficiently complement class I mutant HIV-1. Since HIV-1C130A grew like the wild type, we conclude that Cys-130 is not essential for replication and speculate that perturbation of integrase structure contributed to the pleiotropic HIV-1C130G phenotype.

* 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.

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

{ddagger} Present address: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.

Journal of Virology, December 2004, p. 12735-12746, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.12735-12746.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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