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Journal of Virology, February 2005, p. 1666-1677, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1666-1677.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Recombination of Human Immunodeficiency Virus Type 1 in One Round of Viral Replication: Effects of Genetic Distance, Target Cells, Accessory Genes, and Lack of High Negative Interference in Crossover Events{dagger}

Terence D. Rhodes,1,2 Olga Nikolaitchik,1 Jianbo Chen,1 Douglas Powell,3 and Wei-Shau Hu1*

HIV Drug Resistance Program,1 Data Management Services, Inc., National Cancer Institute at Frederick, Frederick, Maryland,3 Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, West Virginia2

Received 19 May 2004/ Accepted 13 September 2004

Recombination is a major mechanism that generates variation in populations of human immunodeficiency virus type 1 (HIV-1). Mutations that confer replication advantages, such as drug resistance, often cluster within regions of the HIV-1 genome. To explore how efficiently HIV-1 can assort markers separated by short distances, we developed a flow cytometry-based system to study recombination. Two HIV-1-based vectors were generated, one encoding the mouse heat-stable antigen gene and green fluorescent protein gene (GFP), and the other encoding the mouse Thy-1 gene and GFP. We generated derivatives of both vectors that contained nonfunctional GFP inactivated by different mutations. Recombination in the region between the two inactivating mutations during reverse transcription could yield a functional GFP. With this system, we determined that the recombination rates of markers separated by 588, 300, 288, and 103 bp in one round of viral replication are 56, 38, 31, and 12%, respectively, of the theoretical maximum measurable recombination rate. Statistical analyses revealed that at these intervals, recombination rates and marker distances have a near-linear relationship that is part of an overall quadratic fit. Additionally, we examined the segregation of three markers within 600 bp and concluded that HIV-1 crossover events do not exhibit high negative interference. We also examined the effects of target cells and viral accessory proteins on recombination rate. Similar recombination rates were observed when human primary CD4+ T cells and a human T-cell line were used as target cells. We also found equivalent recombination rates in the presence and absence of accessory genes vif, vpr, vpu, and nef. These results illustrate the power of recombination in generating viral population variation and predict the rapid assortment of mutations in the HIV-1 genome in infected individuals.

* Corresponding author. Mailing address: HIV Drug Resistance Program, NCI-Frederick, P. O. Box B, Building 535, Room 336, Frederick, MD 21702. Phone: (301) 846-1250. Fax: (301) 846-6013. E-mail: whu{at}ncifcrf.gov.

{dagger} T.D.R., a medical scientist trainee at West Virginia University, dedicates this article to Mary, Mercy, and Madelyn Rhodes, whose love and sacrifices have helped him further his graduate career.

Journal of Virology, February 2005, p. 1666-1677, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1666-1677.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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