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

To further assess the role of PS, we tested whether various liposomes are able to block hTIM1-mediated viral entry (Fig. 3C ). Consistent with a PS-dependent mechanism, liposomes consisting of 50% PS and 50% PC, but not those consisting of PC alone, efficiently blocked the entry of all hTIM1-using pseudoviruses into hTIM1-expressing 293T cells at 3 mM concentration. In contrast, the entry of LASV and H7N1 pseudoviruses, whose entry is not enhanced by hTIM1, were not affected by either liposomes. Unexpectedly, PE-containing liposomes were also able to inhibit hTIM1-medated viral entry (Fig. S3 ). This observation raises the possibility that PE, another marker of apoptotic cells [52] , which shares structural similarities with PS but is not negatively charged at physiological pH, may also play a role in viral hTIM1 usage. TIM1-mediated virion internalization is independent of viral entry proteins PS dependency of viral hTIM1 usage implies that virions lacking any viral entry protein should also bind to and internalize into the intracellular compartments of hTIM1-expressing cells. To test this we took advantage of GFP-fused matrix proteins [47, 48] that allow, when incorporated into the virions, the detection of prefusion stages of viral entry. As shown in Figures 4A and B , when 293T cells expressing hTIM1, AA-hTIM1 or a control receptor were infected with EBOV VP40-matrix-based VLPs (VP40-GFP VLPs), those bearing no GPs were readily internalized by hTIM1, provided that the TIM1 PSbinding domain was functional. Although EBOV GP-bearing VP40-GFP VLPs appeared to be internalized more efficiently than those lacking GP, this bias is most likely due to the fact that the latter are released 3 to 5 times less efficiently from the producer cells [53, 54] . Consistent with this explanation, when the internalization experiment was repeated with RT-activity normalized MLVgag-GFP virions, we obtained even higher internalization efficiencies for GP-free than EBOV GP-bearing virions (Figs. 4C, D) . Thus, results presented in Figure 4 demonstrate that virions bind to and internalize via hTIM1 in a manner that is independent of specific viral entry proteins, but instead is dependent on components of the viral membrane.