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

In conclusion, our results indicate that hTIM1, hTIM4, hAxl and potentially other PS-binding receptors can enhance the entry of a number of highly divergent viruses. As demonstrated for hTIM1, the enhancement conferred by all of these receptors is likely PS dependent and does not require any viral entry protein. Accordingly, these proteins cannot properly be described as viral receptors, although the nature of the viral entry protein clearly impacts the relevance of PS receptors to infection. In some cases, PS receptors may play critical roles in establishing or maintaining an in vivo infection, which could affect disease severity. Thus our results support the proposal of Soares et al., demonstrated for Pichinde virus and mouse cytomegalovirus [33] , that therapeutic strategies targeting PS and other anionic phospholipids may be broadly effective against a wide range of viruses. Figure S3 PE-containing liposomes also block hTIM1mediated enhancement of viral entry. 293T cells transduced with hACE2 (mock) or hTIM1 were preincubated for 20 min at room temperature with medium alone (none) or with liposomes consisting of either 50% PE and 50% PC (PE/PC) or PC alone (PC). Pseudoviruses or WNV VLPs were then added for a 30 min infection at 37uC, after which unbound liposomes and viruses were washed off and cells supplemented with fresh Figure 9 . A model of viral PS receptor usage. Human TIM proteins (TIMs) and other PS-binding receptors efficiently, but nonspecifically, promote internalization of various enveloped viruses. The TAM receptors Axl, Mer and Tyro (TAMs) similarly bind and internalize many viruses, albeit indirectly via the PS-binding bridging proteins Gas6 and/or Protein S (Prot S) in serum [35] . For most viruses PS-dependent internalization results in a moderate to strong increase of productive infection. Note that the degree of enhancement will depend on the cellular background in which experiments are performed. However, in the case of other viruses, PS receptor-mediated internalization may lead to a compartment that is not productive for infection. In addition, there is a third class of viruses, which is not efficiently bound and/or internalized by PS receptors. The indicated viruses were categorized based on hTIM1 usage and internalization results obtained with pseudoviruses and VLPs (black) or replication-competent viruses (blue). Note, however, that the consequences or efficiency of internalization can vary depending on the PS receptors and their expression levels. doi:10.1371/journal.ppat.1003232.g009 medium. Infection levels were assessed the following day by measuring GFP expression, and normalized to those of untreated hTIM1-expressing cells. Figure shows mean+SD from three independent, duplicated experiments. (EPS) Figure S4 Contribution of stalk length and O-glycans of hTIMs to virus entry. (A) Schematic representation of the three human TIM proteins and stalk-truncated hTIM1 variants used in B-C. hTIM3 differs from the other TIMs because its stalk is shorter and because it has a dramatically reduced number of Oglycans [18] . The two truncation variants of hTIM1, D131-221 and D197-287 hTIM1, were made to resemble hTIM3 in stalk length and number of O-glycosylation sites. (B) Expression levels of stalk-truncated and wt hTIM1 are comparable. 293T cells were transfected with plasmids expressing hACE2 (mock), D131-221 hTIM1, D197-287 hTIM1 or wt hTIM1. 48 h later cells were stained with the anti-hTIM1 antibody 3D1, which