GHOST Cell Infection Assay for Viral Coreceptor Usage

The GHOST cell infection assay was used to determine coreceptor usage of viral stocks. Parental, CXCR4-expressing, or CCR5-expressing GHOST cells were cultured in 24-well plates. The cells were infected with undiluted PSV in the presence of 20 μg/mL of Polybrene infection reagent (Millipore-Sigma, Burlington, MA, USA) for 4 h. The cells were washed, cultured for 2 days, and then harvested and fixed with 2% paraformaldehyde. The percentage of infected cells expressing green fluorescent protein (GFP) was measured by flow cytometry using a LSRII cytometer and FACSDIVA software (Becton, Dickinson, Franklin Lakes, NJ, USA). Postgating analysis was conducted with FlowJo software (FlowJo, LLC, Ashland, OR, USA). PSVs were designated using the CCR5 or CXCR4 receptor if the percentage of GFP-positive cells for either the CCR5- or CXCR4- cell lines was 5-fold greater than the percentage of GFP-positive cells in the parental line. Assay controls included an uninfected negative-control, murine leukemia virus (MuLV) as a positive control for all lines and BaL.01 (CCR5-tropic) and MN.3 (CXCR4-tropic) PSVs as specific cell line controls.