HIV cell-to-cell spread slows evolution of drug resistance

Here we investigated whether a combination of cell-free infection and cell-to-cell spread confers a selective advantage in the evolution of resistance to an inhibitor relative to cell-to-cell spread alone due to the stronger selection pressure against drug sensitive virus. We propagated HIV infection using coculture of infected with uninfected cells in the face of the reverse transcriptase inhibitor efavirenz (EFV), and compared the effect on drug resistance evolution of including one cycle of cell-free infection. In the presence of a single cell-free infection step, we obtained earlier evolution of resistance to EFV. When we increased selective pressure by adding emtricitabine (FTC as a second drug, infection with the cell-free step evolved multidrug resistance and was able to replicate, while infection without a cell-free step failed to evolve multidrug resistance. In conclusion, our results suggest that, HIV cell-to-cell spread has a decreased capacity to rapidly evolve resistance to inhibitors, which is conferred by cell-free infection. Mathenmatical modeling of evoltion of drug resistance by cell-free infection and cell-to-cell sread supported by sequencing of Reverse transciptase region of proviral DNA from infected cell populations over 48hr timepoints at 20nM EFV, 40nM EFV and a combination of 20nM EFV and 770nM FTC E7 clone was generated by subcloning RevCEM cells at single cell density. Surviving clones were subdivided into replicate plates. One of the plates was screened for the fraction of GFP expressing cells upon HIV infection using microscopy, and the clone with the highest fraction of GFP positive cells was selected