TIM-family Proteins Promote Infection of Multiple Enveloped Viruses through Virion-associated Phosphatidylserine

Jemielity, Stephanie; Wang, Jinyize J.; Chan, Ying Kai; Ahmed, Asim A.; Li, Wenhui; Monahan, Sheena; Bu, Xia; Farzan, Michael; Freeman, Gordon J.; Umetsu, Dale T.; DeKruyff, Rosemarie H.; Choe, Hyeryun

VLPs made with EBOV VP40 matrix proteins fused to blactamase (Bla) and bearing EBOV GP, LASV GP or no GP were produced as described above for VP40-GFP VLPs. The VP40-Bla fusion construct was a gift from Paul Bates at University of Pennsylvania [8] . Naive peritoneal macrophages were obtained from wildtype BALB/cBYJ mice. Briefly, peritoneal cavity cells were plated in 12-well plates at 10 6 per well and incubated for 1 h at room temperature to let macrophages adhere. After removing non-adherent cells by washing twice in PBS/2% FBS, macrophages were incubated overnight at 37uC in RPMI containing 10% FBS, 1% P/S and 50 mM b-mercaptoethanol and infected the following day for 2 h at 37uC with VP40-Bla VLPs. Infected cells were detached by scraping in trypsin/EDTA, washed and loaded with the Bla substrate CCF2-AM, as previously described [49] . The conversion of substrate by cytoplasmic esterases and Bla, which reflects VP40-Bla VLP entry, was detected using the LSR II flow cytometer (BD Biosciences).