Background. T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) 28 receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 29 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains 30 unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have 31 suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a 32 BSL2 model virus that expresses EBOV glycoprotein and demonstrate the importance of TIM-1 33 as a virus receptor late during in vivo infection. 34 Methodology/Principal findings. We used an infectious, recombinant vesicular stomatitis virus 35 expressing EBOV glycoprotein (EBOV GP/rVSV) to assess the role of TIM-1 during in vivo 36 infection. TIM-1-sufficient or TIM-1-deficient BALB/c interferon α/β receptor -/mice were 37 challenged with EBOV GP/rVSV-GFP or G/rVSV-GFP. While G/rVSV caused profound 38 morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better 39 survival than TIM-1-expressing mice following EBOV GP/rVSV challenge. EBOV GP/rVSV 40 load in spleen was high and unaffected by expression of TIM-1. However, infectious virus in 41 serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, 42 suggesting that virus entry via this receptor contributes to virus load. Consistent with higher 43 virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-44 sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more 45 modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were 46 depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. 47 Depletion of T cells did not alter the pathogenic consequences of virus infection. 48 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/466102 doi: bioRxiv preprint Conclusions. Our studies provide evidence that at late times during EBOV GP/rVSV infection, 49 TIM-1 increased virus load and associated mortality, consistent with an important role of this 50 receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction 51 may serve as effective antivirals, reducing virus load at late times during EBOV infection. 52 53 Author summary 54 T cell immunoglobulin mucin domain-1 (TIM-1) is one of a number of phosphatidylserine (PS) 55 receptors that mediate clearance of apoptotic bodies by binding PS on the surface of dead or 56 dying cells. Enveloped viruses mimic apoptotic bodies by exposing PS on the outer leaflet of the 57 viral membrane. While TIM-1 has been shown to serve as an adherence factor/receptor for 58 filoviruses in tissue culture, limited studies have investigated the role of TIM-1 as a receptor in 59 vivo. Here, we sought to determine if TIM-1 was critical for Ebola virus glycoprotein-mediated 60 infection using a BSL2 model virus. We demonstrate that loss of TIM-1 expression results in 61 decreased virus load late during infection and significantly reduced virus-elicited mortality. 62 These findings provide evidence that TIM-1 serves as an important receptor for Ebola virus in 63 vivo. Blocking TIM-1/EBOV interactions may be effective antiviral strategy to reduce viral load 64 and pathogenicity at late times of EBOV infection. 65 66 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/466102 doi: bioRxiv preprint 129 for maEBOV we observed that EBOV GP/rVSV was less pathogenic in TIM-1-deficient mice 130 compared to control mice. The impact of the loss of TIM-1 was specific for EBOV GP-131 expressing virus since wild-type VSV was equally virulent in TIM-1-deficient and TIM-1-132 sufficient mice over a wide range of challenge doses. Importantly, reduced mortality observed 133 in the virus-infected TIM-1 -/mice was associated with lower virus load at late time points 134 during infection in multiple tissues previously appreciated to be important in EBOV 135 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/466102 doi: bioRxiv preprint 157 Havcr1, were replaced with a LacZ gene, generating a TIM-1-null mouse (TIM-1 -/-) BALB/c 158 IFN-αβ receptor-deficient (Ifnar -/-) mice were a kind gift from Dr. Joan Durbin, NYU Langone 159 . CC-BY 4.0 International license is made available under a
Output: 2 3 TIM-1 SERVES AS A NONREDUNDANT RECEPTOR FOR EBOLA 4 VIRUS, ENHANCING VIREMIA AND PATHOGENESIS 5 6