E-MTAB-8357
MiSeq 2x300 paired-end sequencing of HEK293T cells stably expressing eGFP-PPP2R5A transduced with a HIV-1 Vif mutant library
The goal of this experiment was to map the binding surface between the HIV-1 Vif protein and the cellular phospho-regulator PPP2R5A. Previous studies had shown that Vif can target PPP2R5A for proteasomal degradation but the binding surface required had yet to be elucidated. For this experiment, HEK293T cells stably expressing eGFP-PPP2R5A were transduced with virus containing a Vif mutant library. The transduction was done at a low multiplicity of infection so that, on average, each cell only received a single Vif variant. The Vif library was built using a mCherry-T2A-Vif expression cassette so transduced cells could be monitored via mCherry fluorescence. Once the library had been introduced into the eGFP-PPP2R5A cells, fluorescence-activated cell sorting was used to separate out transduced cells that could, and could not, degrade eGFP-PPP2R5A. For example, if a cell received a functional variant, it would lose eGFP fluorescence (PPP2R5A degradation) but retain mCherry fluorescence. On the other hand, if a variant was non-functional, eGFP fluorescence would persist (no PPP2R5A degradation). The unsorted transduced population, mCherry+/eGFP-, and mCherry+/eGFP+ populations were subjected to a 2x300 bp MiSeq run to comprehensively identify mutants that were functional and non-functional.
The goal of this experiment was to map the binding surface between the HIV-1 Vif protein and the cellular phospho-regulator PPP2R5A. Previous studies had shown that Vif can target PPP2R5A for proteasomal degradation but the binding surface required had yet to be elucidated. For this experiment, HEK293T cells stably expressing eGFP-PPP2R5A were transduced with virus containing a Vif mutant library. The transduction was done at a low multiplicity of infection so that, on average, each cell only received a single Vif variant. The Vif library was built using a mCherry-T2A-Vif expression cassette so transduced cells could be monitored via mCherry fluorescence. Once the library had been introduced into the eGFP-PPP2R5A cells, fluorescence-activated cell sorting was used to separate out transduced cells that could, and could not, degrade eGFP-PPP2R5A. For example, if a cell received a functional variant, it would lose eGFP fluorescence (PPP2R5A degradation) but retain mCherry fluorescence. On the other hand, if a variant was non-functional, eGFP fluorescence would persist (no PPP2R5A degradation). The unsorted transduced population, mCherry+/eGFP-, and mCherry+/eGFP+ populations were subjected to a 2x300 bp MiSeq run to comprehensively identify mutants that were functional and non-functional.