Enhanced isolation of low frequency herpes simplex virus recombinants using green-fluorescent protein and FACS

The generation of recombinant herpes simplex virus to study the effect of engineered mutations on viral biology relies on the isolation of recombinants from a mixed population of viruses following a marker transfer procedure. Currently, the E. coli lacZ or green-fluorescent protein (GFP) genes are most frequently used as markers for isolation and isolation of recombinants relies on visual screening of plaques. Alternatively, novel restriction site changes can be inserted into a gene followed by screening of individual plaques for the novel change. These methods are inefficient when the frequency of recombinants in the pool of viruses is low. Using GFP as a selection marker, a FACS procedure that results in a substantial enrichment of the frequency of recombinants is described. Cells were infected at a multiplicity of infection (MOI) of 1.0 in the presence of acyclovir and at 10h post-infection, either the GFP+ or GFP- cells were sorted by FACS, and the sorted cells were plated on fresh cells. After three rounds of selection, the frequency of GFP+ recombinants rose from 0.1 to 3-4%. A mutant virus with a GFP insertion in the US1 gene (alpha22 protein) was generated and then used to isolate a virus with a mutation, Y116C, in the alpha22 protein.