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

Lyophilized infectious TCRV (TRVL 11573), passaged in suckling mice and Vero cells, as well as RRV (T-48) and SINV (Ar-339), both passaged in suckling mice, were purchased from ATCC, and resuspended in PBS according to the instruction. Type 2 infectious dengue virus (DENV, New Guinea C) was obtained by passaging in C6/36 Aedes albopictus mosquito cells. Virus-containing culture supernatants were 0.45 mm-filtered, stored at 280uC and virus titers determined using plaque assays in baby hamster kidney cells as described [44] . Infectious FLUAV (H1N1, A/PR/8/34) was propagated in MDCK cells [45] , 0.45 mm-filtered and virus titers assessed as tissue culture infectious dose 50 (TCID50) in cultured human macrophages using CellTiter-Glo One solution (Promega). For infection, cells were incubated for 1-6 h at 37uC with viruses serially diluted in DMEM containing 10% FBS, washed and supplemented with fresh medium. At indicated days post-infection, cells were detached by trypsinization, washed, fixed with 1% formaldehyde in PBS and permeabilized with 0.1% saponin in PBS containing 2% goat serum. Cells were then stained with immune ascitic fluids (ATCC) for TCRV, RRV and SINV, anti-FLUAV antibody C111 (Clontech), or anti-DENV antibody 2H2 (Millipore) [46] followed by an Alexa 649-or PE-conjugated goat anti-mIgG antibody (Jackson ImmunoResearch Laboratories) and analyzed by flow cytometry.