Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry

T-cell immunoglobulin andmucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidyl-serine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent inter-nalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indi-cating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhance-ment of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasmamembrane bound. Based on these defined char-acteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of -dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chi-kungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated en-hancement of virus entry provides a basis for more effective recognition of unknown PVEERs