Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 10;90(13):6097-6111.
doi: 10.1128/JVI.00100-16. Print 2016 Jul 1.

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

Bethany A Rhein  1 , Rachel B Brouillette  1 , Grace A Schaack  1 , John A Chiorini  2 , Wendy Maury  3
Affiliations

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

Bethany A Rhein et al. J Virol. .

Abstract

Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion-TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions.

Importance: With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Expression of human or murine TIM-4 enhances EBOV entry. (A) EBOV entry was enhanced when increasing amounts of hTIM-4 and mTIM-4 were transfected into HEK 293T cells. Forty-eight hours following transfection, cells were transduced with EBOV VSVΔG-GFP pseudovirions. Twenty-four hours later, transduction was quantified by assessing GFP expression by flow cytometry. Background transduction of empty-vector-transfected HEK 293T is indicated by the dotted line (∼1.5% of cells). (B) Amino acid sequence alignment of the mTIM-4 and hTIM-4 IgV domains generated with the Clustal W software program. β-Sheets are indicated by a line and a corresponding letter above the alignment, as previously assigned by Santiago et al. (25). Residue numbers are noted below the amino acid sequences. Asterisks represent conserved sequences, colons represent conservative mutations, and periods represent semiconservative mutations. (C) Overlay of the IgV domain structures of mTIM-4 (blue) and hTIM-4 (green). IgV domain structures of mTIM-4 (PDB code 3BI9) (25) and hTIM-4 (PDB code 5DZN) (28) were obtained from the RCSB PDB and aligned in PyMOL. (D) Residues found in this study to impact EBOV entry are identified in the structures of the mTIM-4 IgV domain (left) and the hTIM-4 IgV domain (right). β-Sheets (mTIM-4, blue; hTIM-4, green) are labeled with the corresponding letters. Red residues are located in the PtdSer binding pocket, and teal residues are located outside the PtdSer binding pocket.
FIG 2
FIG 2
TIM-4 IgV domain PtdSer binding pocket residues N121 and D122 are critical for EBOV entry. HEK 293T cells were transfected with WT or N121/D122 TIM-4 IgV domain mutant constructs and assessed for their impact on EBOV VSVΔG-GFP pseudovirion transduction (A) and EBOV GP/rVSV infection (B). GFP expression was quantified by flow cytometry 24 h following transduction or infection. Relative transduction and infection were normalized to TIM-4 surface expression for each construct as described in Materials and Methods. WT TIM-4 represents 100% transduction and infection in panels A and B, respectively. Significance was determined by one-sample t test. **, P < 0.01; ***, P < 0.001. (C) ND121/122DN (DN) mutant TIM-4 does not enhance EBOV GP/rVSV infection. Forty-eight hours following transfection with TIM-4 plasmids or the empty vector, cells were infected with serial dilutions of EBOV GP/rVSV. Infection was quantified by flow cytometry 48 h following infection. (D) ND mutant TIM-4 constructs have reduced binding to EBOV VSVΔG-GFP pseudovirions. HEK 293T supernatants containing HA-tagged WT or mutant TIM-4 protein were incubated on ELISA plates prebound with EBOV VSVΔG-GFP pseudovirions. Relative protein amounts present in supernatants in a representative immunoblot assay are shown at the bottom. The dotted line represents TIM-4 binding to plates not coated with pseudovirions. Soluble TIM-4 proteins were detected with anti-HA antiserum. Significance was determined by Student t test compared to WT TIM-4 binding to EBOV pseudovirions. ***, P < 0.001. (E) WT TIM-4, not the DN PtdSer binding pocket mutant, increases internalization of FITC-labeled EBOV VSVΔG-GFP pseudovirions. HEK 293T cells were transfected with WT TIM-4 or ND121/122DN mutant TIM-4. Pseudovirions were bound at 4°C for 1 h and then shifted to 37°C for the time indicated to allow virus internalization. Cells were treated with trypsin-EDTA to remove cell surface-bound, noninternalized virus. FITC expression was quantified by flow cytometry following trypsin-EDTA treatment and washing. Internalization into empty-vector-transfected cells represents 100%. Significance was determined by one-sample t test. **, P < 0.01; ***, P < 0.001.
FIG 3
FIG 3
Additional PtdSer binding pocket hTIM-4 IgV domain residues affect EBOV entry. HEK 293T cells were transfected with WT or mutant TIM-4 constructs and assessed for their impact on EBOV VSVΔG-GFP pseudovirion transduction (A) or EBOV GP/rVSV infection (B). GFP expression was quantified by flow cytometry 24 h following transduction or infection. Relative transduction and infection were normalized to TIM-4 surface expression for each construct as described in Materials and Methods. WT TIM-4 represents 100% transduction and infection in panels A and B, respectively. The significance of differences from WT TIM-4 was determined by one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Y61A and S62A mutant hTIM-4 CC″ loop constructs demonstrated about 4-fold less infectivity than WT hTIM-4. Forty-eight hours following transfection with TIM-4 plasmids or the empty vector, cells were infected with serial dilutions of EBOV GP/rVSV. Infection was quantified by flow cytometry 48 h following infection. The significance of differences from WT TIM-4 infection was determined by Student t test. **, P < 0.01. (D) Identification of TIM-4 PtdSer binding pocket mutant constructs with reduced binding to EBOV VSVΔG-GFP pseudovirions. HEK 293T supernatants containing HA-tagged WT or mutant TIM-4 proteins were incubated on ELISA plates prebound with EBOV VSVΔG-GFP pseudovirions. The dashed line represents binding of WT pseudovirions to an uncoated plate. Relative soluble TIM-4 expression present in supernatants is shown below in a representative immunoblot assay with anti-HA antiserum. The significance of differences from WT TIM-4 binding to EBOV pseudovirions was determined by Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 4
FIG 4
More FG loop residues are required for optimal hTIM-4-mediated entry than for mTIM-4-mediated entry. HEK 293T cells were transfected with WT TIM-4 or FG loop mutant constructs and assessed for their impact on EBOV VSVΔG-GFP pseudovirion transduction (A) and EBOV GP/rVSV infection (B). GFP expression was quantified by flow cytometry 24 h following transduction or infection. Relative transduction and infection were normalized to TIM-4 surface expression for each construct as described in Materials and Methods. WT TIM-4 represents 100% transduction and infection in panels A and B, respectively. The significance of differences from WT TIM-4 was determined by one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) FG loop mutant TIM-4 constructs with reduced transduction and infection also have reduced binding to EBOV VSVΔG-GFP pseudovirions. HEK 293T supernatants containing HA-tagged WT or mutant TIM-4 protein were incubated with ELISA plates prebound with EBOV VSVΔG-GFP pseudovirions. The dashed line represents the binding of WT pseudovirions to an uncoated plate. Relative amounts of soluble TIM-4 present in supernatants in a representative immunoblot assay with anti-HA antiserum are shown at the bottom. The significance of differences from WT TIM-4 binding to EBOV pseudovirions was determined by Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
TIM-4 IgV domain residues outside the PtdSer binding pocket are important for EBOV entry. HEK 293T cells were transfected with WT or IgV domain mutant TIM-4 constructs outside the PtdSer binding pocket and assessed for their impact on EBOV VSVΔG-GFP pseudovirion transduction (A) and EBOV GP/rVSV infection (B). GFP expression was quantified by flow cytometry 24 h following transduction or infection. Relative transduction and infection were normalized to TIM-4 surface expression for each construct as described in Materials and Methods. WT TIM-4 represents 100% transduction and infection in panels A and B, respectively. The significance of differences from WT TIM-4 was determined by one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) hTIM-4 residues outside the PtdSer binding pocket with reduced binding to EBOV VSVΔG-GFP pseudovirions were identified. HEK 293T supernatants containing HA-tagged WT or mutant TIM-4 proteins were incubated on ELISA plates prebound with EBOV VSVΔG-GFP pseudovirions. The dashed line represents the binding of WT pseudovirions to an uncoated plate. Relative protein amounts present in supernatants in a representative immunoblot assay with anti-HA antiserum are shown at the bottom. The significance of differences from WT TIM-4 binding to EBOV pseudovirions was determined by Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 6
FIG 6
mTIM-4 and hTIM-4 IgV domain mutants found to inhibit EBOV entry also have reduced binding to PtdSer liposomes. HEK 293T supernatants containing HA-tagged mouse (A) or human (B) WT or mutant TIM-4 proteins were incubated on ELISA plates prebound with 50 μM PtdSer or PtdC liposomes. Relative protein amounts present in supernatants were normalized in an immunoblot assay with anti-HA antiserum prior to ELISA analysis. The significance of differences from WT TIM-4 protein PtdSer binding was determined by Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
FIG 7
FIG 7
TIM-4 IgV RGD motifs are not important for EBOV entry. (A) HEK 293T cells express integrin αv but not Mer. Polyclonal antiserum (black histogram) against human integrin αv or Mer and a control goat IgG (gray histogram) were used to stain the surface of HEK 293T cells. Surface expression was analyzed by flow cytometry. (B) Mutations in the mTIM-4 IgV domain RGD motif do not reduce EBOV VSVΔG-GFP pseudovirion transduction. HEK 293T cells were transfected with WT or mutant TIM-4 constructs. Transfected cells were transduced with EBOV VSVΔG-GFP pseudovirions at 24 h. GFP expression was quantified 24 h following transduction by flow cytometry. WT mTIM-4 represents 100% transduction, and significance was determined by one-sample t test. ***, P < 0.001. (B) hTIM-4-mediated EBOV entry is not blocked by increasing concentrations of RGD peptide. HEK 293 cells stably expressing hTIM-4 were preincubated with increasing concentrations of RGD peptide or the EBOV entry inhibitor 3.47 at 37°C for 30 min. Cells were transduced in the presence of the peptide or inhibitor with FL EBOV VSVΔG-GFP pseudovirions, and GFP expression was quantified by flow cytometry 24 h following transduction. Transduction of cells in medium alone represents 100%.
FIG 8
FIG 8
Identification of TIM-1 and TIM-4 residues important for filovirus entry. (A) Amino acid sequence alignment of the hTIM-1, hTIM-4, and mTIM-4 IgV domains generated with the Clustal W software program. Each β-sheet is indicated above the alignment by a line and a corresponding letter, as previously assigned by Santiago et al. (25). Residue numbers are below the amino acid sequences. Asterisks represent conserved sequences, colons represent conservative mutations, and periods represent semiconservative mutations. (B) Overlay of the IgV domain structures of hTIM-1 (gray) and hTIM-4 (black). The IgV domain structures of hTIM-1 (PDB code 5DZO) and hTIM-4 (PDB code 5DZN) (28) were obtained from the RCSB PDB and aligned in PyMOL. Highlighted residues indicate those found in this study and that of Moller-Tank et al. (12) to impact EBOV entry. Purple (hTIM-1) and cyan (hTIM-4) residues are important for both TIM molecules. Green residues are uniquely important for hTIM-4.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Murray MJ. 2015. Ebola virus disease: a review of its past and present. Anesth Analg 121:798–809. doi:10.1213/ANE.0000000000000866. - DOI - PubMed
    1. WHO. 2015. Ebola situation reports. World Health Organization, Geneva, Switzerland: http://apps.who.int/ebola/ebola-situation-reports Accessed 9 September 2015.
    1. Martines RB, Ng DL, Greer PW, Rollin PE, Zaki SR. 2015. Tissue and cellular tropism, pathology and pathogenesis of Ebola and Marburg viruses. J Pathol 235:153–174. doi:10.1002/path.4456. - DOI - PubMed
    1. Rhein B, Maury W. 2015. Ebola virus entry into host cells: identifying therapeutic strategies. Curr Clin Microbiol Rep 2:115–124. doi:10.1007/s40588-015-0021-3. - DOI - PMC - PubMed
    1. Alvarez CP, Lasala F, Carrillo J, Muniz O, Corbi AL, Delgado R. 2002. C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J Virol 76:6841–6844. doi:10.1128/JVI.76.13.6841-6844.2002. - DOI - PMC - PubMed

Publication types

MeSH terms