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Background:  Quantitative models of HIV-1 dynamics, diversification and escape need to account for the contribution of genetic recombination but have yet to do so. Important aspects of HIV-1 recombination remain poorly defined, including the propensity of HIV-1 to productively infect cells with more than one virion (a necessary precondition for recombinational diversification) and the frequency of recombination events during infection of the natural target cell population. Because prior methods for analysis of recombination in tissue culture have employed antibiotic drug resistance markers to select and enumerate infected cells, these studies have been limited to single round infection of fibroblastic cell lines.

Methods:  We have developed methods using HIV-1 reporter viruses bearing genes for Green, Yellow and Cyan Fluorescent Proteins which allows immediate and simultaneous quantification by flow cytometry of the rate of virus infection, co-infection and recombination within any cell type, including primary T cells and macrophages as well as the SCID-hu (Thy/Liv) mouse model.

Results:  Single round infection assays reveal that HIV-1 is up to an order of magnitude more recombinogenic that previously observed in fibroblasts, with infection of CD4⁺ T cells generating an average of 9 recombination events per virus, and infection of macrophages producing about 30.  Differentiation of two monocytic cell lines, THP-1 and U937 leads to a 3-fold enhancement of recombination rates, from levels similar to T cells up to the levels observed in primary macrophages, indicating that myeloid differentiation elicits or modulates the expression of cellular factor(s) which interact with the HIV-1 reverse transcription process and influence recombination.

Conclusions:  Multiround infection assays reveal the dynamics of HIV-1 co-infection and recombination. Co-infection of cells is subject to little functional inhibition, despite the known ability of HIV-1 to down modulate the cellular CD4 receptor. As a result, the generation of recombinants proceeds according to the square of the infection frequency and is independent of input MOI, the number of rounds of virus replication and cell donor. Identical results are obtained in both the tissue culture and SCID-Hu systems, suggesting that generally applicable relationships are described. If operative in human infection, these results suggest that increases in viral load may confer a compounding increased risk of virus escape via recombinational diversification.

Keywords: Recombination; Diversification; SCID-hu