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Journal of Virology, October 2003, p. 11050-11059, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11050-11059.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Replication of Chimeric Human Immunodeficiency Virus Type 1 (HIV-1) Containing HIV-2 Integrase (IN): Naturally Selected Mutations in IN Augment DNA Synthesis

Marcus Padow,1 Lilin Lai,2 Champion Deivanayagam,3 Lawrence J. DeLucas,3 Robert B. Weiss,4 Diane M. Dunn,4 Xiaoyun Wu,2 and John C. Kappes1,2,5*

Departments of Microbiology,1 Medicine,2 Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35294,3 Department of Genetics, University of Utah, Salt Lake City, Utah 84112,4 Birmingham Veterans Affairs Medical Center Research Services, Birmingham, Alabama 352335

Received 8 April 2003/ Accepted 16 July 2003

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein augments the initiation of reverse transcription. Chimeric HIV-1 containing HIV-2 IN (SG3IN2) is severely impaired in virus infectivity and DNA synthesis. To analyze the nature of this defect, we infected T cells with the chimeric SG3IN2 virus and by continuous passage in cell culture selected for virus with improved replication properties. Viruses from two different time points were chosen for further analysis, an early culture-adapted virus (CF-65) that exhibited an intermediate level of infectivity, and a later-passaged virus (CF-131) that was significantly more infectious. Sequence analysis of multiple clones derived from the CF-65 virus culture demonstrated a diversity of mutations in the reverse transcriptase (RT) and a common V204I IN mutation. Analysis of clones derived from the CF-131 virus indicated the selection of two additional IN mutations, Q96H and K127E, and a fixed V179I RT mutation. By cloning RT and/or IN sequences back into the original SG3IN2 chimeric virus, we demonstrated that mutations in both RT and IN contributed to the improvement in viral fitness. The effect of the HIV-2IN(IN2) mutations on virus DNA synthesis was analyzed by packaging IN2 mutants into HIV-1 as Vpr-IN2 fusion proteins. This analysis revealed that the Q96H, K127E and V204I mutations increased the infectivity of the chimeric virus by augmenting the initiation of viral cDNA synthesis in infected cells. The Q96H and K127E mutations are present in adjacent helical structures on the surface of the IN protein and together account for most of the increase observed in DNA synthesis. Our findings provide evidence that the IN protein augments the initiation of reverse transcription through specific interactions with other viral components comprising the initiation complex. Moreover, they implicate specific regions on the surface of IN that may help to elucidate mechanisms by which the HIV-1 IN protein augments the initiation of HIV-1 reverse transcription in vivo.

* Corresponding author. Mailing address: University of Alabama at Birmingham, 701 19th St. South, Birmingham, AL 35294. Phone: (205) 934-0051. Fax: (205) 975-7300. E-mail: kappesjc{at}uab.edu.

Journal of Virology, October 2003, p. 11050-11059, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11050-11059.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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