Ebola viruses, and other members of the family filoviridae, are enveloped, negative sense, RNA viruses that can cause hemorrhagic fever. Currently, there are no antivirals or approved vaccines available that target or protect from Ebola virus infection. However, recently, T-cell immunoglobulin and mucin domain-1 (TIM-1) has been identified as an epithelial-cell receptor for filoviruses and could be a potential target for antivirals. However, little is known about how TIM-1 enhances virus entry and the role of TIM-1 during in vivo infection. In order to determine the key residues of TM-1 involved in interaction with virus, we generated a panel of point-mutations in the immunoglobulin-like variable (IgV) domain of TIM-1. We determined that several residues within the IgV domain that are involved in binding of phosphatidylserine (PtdSer) are also critical for Ebola virus entry. Further, we found that TIM-1 interacts with Ebola virus through binding of PtdSer on the viral envelope. PtdSer liposomes, but not phosphatidylcholine liposomes, competed with TIM-1 for EBOV pseudovirion binding and transduction. In addition, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. We also determined that TIM-1 expression can enhance infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped virion associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like particles (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped virus uptake. The PtdSer-binding activity of the IgV domain is essential for both virus binding and internalization by TIM-1. However, another member of the TIM family, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry. These data indicate that other domains of TIM proteins are functionally important. We investigated the domains of the TIM family members that play a role in the enhancement 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 plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. Provided that expression of TIM-1 in cells enhances virus entry through binding of PtdSer on the viral membrane, we wanted to determine whether virus entry would still be enhanced if this interaction was reversed with TIM-1 present on the viral membrane. Further, we reasoned that this might allow for targeting of virus to cells with greater amounts of PtdSer exposed on their outer leaflet, such as cancer cells. In order to test this hypothesis, we generated virions in cells coexpressing a glycoprotein and one of the TIM family members. We found that expression of TIMs in virus-producing cells resulted in TIM proteins being released into the virus-containing medium and enhanced Ebola virus GP pseudovirion titers. Further, this enhancement was dependent on the amount of PtdSer exposed on the target-cell membrane. However, we also determined that TIMs were not being incorporated into virions and that coexpression of TIMs with non-ebolavirus glycoproteins in virus-producing cells resulted in virus stocks with both reduced titers and the quantity of virions.

Les polérovirus infectent une large gamme de plantes d’intérêt économique. Ils sont transmis par un insecte vecteur, le puceron, selon le mode circulant non-multipliant. Le virus, acquis par le puceron lors de l’ingestion de sève sur une plante infectée, traverse l’épithélium des cellules intestinales puis celui des glandes salivaires par un mécanisme de transcytose impliquant des récepteurs encore inconnus. Le récepteur de l’éphrine (Eph) est une protéine membranaire dont un domaine est capable de se lier dans la levure aux protéines structurales des polérovirus. En développant des techniques basées sur l’ARN interférence, nous avons montré que l’acquisition orale d’ARN double brin ciblant Eph chez le puceron Myzus persicae permet de réduire de manière reproductible l’internalisation des polérovirus dans le corps du puceron. Les pucerons ainsi traités transmettent le virus avec une efficacité réduite. Eph pourrait donc assurer la fonction de récepteur des polérovirus chez M. persicae. / Poleroviruses infect a wide range of economically important plants. They are transmitted in a circulative and non-propagative mode by an insect vector, the aphid. The virus particles are acquired by aphids when ingesting the sap from an infected plant and cross successively the epithelia of the midgut and the salivary gland cells by a transcytosis mechanism that relies on the presence of unknown receptors.The ephrin receptor (Eph) is a membrane protein which contains a domain able to bind in yeast to the structural proteins of poleroviruses. By developing methods based on RNA interference, we have shown that oral acquisition of double-stranded RNA targeting Eph in the aphid Myzus persicae can reproducibly reduce polerovirus internalization into the aphid's body. Such treated aphids transmit the virus to plants with a lower efficiency. Eph could therefore function as a receptor for poleroviruses in M. persicae.