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

Pseudoviruses bearing various viral entry proteins were produced in 293T cells as described [39] by calcium-phosphate transfection of a retroviral vector pQCXIX (BD Biosciences) encoding eGFP together with two other plasmids, separately encoding a viral entry protein and the Moloney murine leukemia virus (MLV) gag and pol. For FLUAV H7N1, an additional plasmid encoding N1 neuraminidase (Puerto Rico) was cotransfected [41] . Pseudovirus-containing culture supernatants were harvested at 32-34 h post-transfection, filtered through a 0.45 mm PES membrane, stored at 4uC, and used within 2 weeks. WNV VLPs were produced and harvested in the same way after cotransfecting a plasmid encoding the WNV structural proteins (capsid and entry glycoproteins prM and E) and a WNV replicon encoding the non-structural proteins NS1-5 and GFP [43] .