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
. 2005 Sep 30;340(2):224-36.
doi: 10.1016/j.virol.2005.06.026.

LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus

Thomas Gramberg  1 , Heike Hofmann, Peggy Möller, Patricia F Lalor, Andrea Marzi, Martina Geier, Mandy Krumbiegel, Thomas Winkler, Frank Kirchhoff, David H Adams, Stephan Becker, Jan Münch, Stefan Pöhlmann
Affiliations

LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus

Thomas Gramberg et al. Virology. .

Abstract

Cellular attachment factors like the C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can augment viral infection and might promote viral dissemination in and between hosts. The lectin LSECtin is encoded in the same chromosomal locus as DC-SIGN/R and is coexpressed with DC-SIGNR on sinusoidal endothelial cells in liver and lymphnodes. Here, we show that LSECtin enhances infection driven by filovirus glycoproteins (GP) and the S protein of SARS coronavirus, but does not interact with human immunodeficiency virus type-1 and hepatitis C virus envelope proteins. Ligand binding to LSECtin was inhibited by EGTA but not by mannan, suggesting that LSECtin unlike DC-SIGN/R does not recognize high-mannose glycans on viral GPs. Finally, we demonstrate that LSECtin is N-linked glycosylated and that glycosylation is required for cell surface expression. In summary, we identified LSECtin as an attachment factor that in conjunction with DC-SIGNR might concentrate viral pathogens in liver and lymph nodes.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Inducible expression of LSECtin on 293 T-REx cells and endogenous expression on LSEC. (A–E) Regulated LSECtin expression on cell lines. Cell lines were generated by stable transfection and lectin expression was analyzed with an antiserum raised against a GST-LSECtin fusion protein (A–D) or with pre-immune serum (E). Lectin expression on doxycycline (dox)-induced LSECtin T-REx cells (black) was compared to the following cells (white): (A) uninduced LSECtin T-REx cells, (B and E) induced T-REx control cells, (C) induced DC-SIGN T-REx cells, (D) induced DC-SIGNR T-REx cells. (F) Expression of LSECtin on LSECs. Primary human LSECs were obtained from human liver tissue and analyzed for LSECtin expression as described above. Similar results were obtained with cells from three different donors.
Fig. 2
Fig. 2
Impact of LSECtin expression on viral infection. (A) LSECtin and DC-SIGN/R augment infection by lentiviral pseudotypes bearing filovirus GPs or SARS-CoV-S. Lentiviral pseudotypes harboring the GPs of the indicated viruses were normalized for comparable infection of 293T control cells and used to infect 293T cells transiently expressing DC-SIGN/R, LSECtin, or empty vector. The reporter viruses employed contain the luciferase gene, which is expressed only upon integration of the proviral DNA into the cellular genome. Three days after infection, the cells were lysed and luciferase activity was determined. Infection is shown relative to infectious entry into control cells, which was set as 100%. Upon infection of control cells with ZEBOV-GP, SARS-CoV-S, or VSV-G bearing pseudotypes, luciferase activities of 9049 ± 1546, 19,810 ± 6809, and 6341 ± 676 counts per second (c.p.s.) were measured. The result of a representative experiment is shown, similar results were obtained in three independent experiments. Error bars indicate standard deviation (SD). (B) LSECtin expression does not allow SARS-CoV-S driven infection of non-permissive cells. HeLa cells, which do not express the SARS-CoV receptor ACE2 and are refractory to SARS-CoV-S driven infection, were transiently transfected with LSECtin or ACE2 or cotransfected with both plasmids. The transfected cells were subsequently infected with SARS-CoV-S bearing pseudotypes and luciferase activity was determined as described under panel A. The results of a representative experiment performed in quadruplicates are shown, error bars indicate SD. Similar results were obtained in two independent experiments. (C) Inhibition of infection by LSECtin-specific antiserum. T-REx cell lines were induced to express DC-SIGN or LSECtin, incubated with LSECtin-specific antiserum and infected with ZEBOV-GP harboring pseudotypes, which were normalized for equal luciferase activity upon infection of control cells. A representative experiment is shown, error bars indicate SD. Similar results were obtained in an independent experiment.
Fig. 3
Fig. 3
LSECtin does not interact with HIV-1 and infectious pseudovirions bearing the HCV E1 and E2 proteins. (A) Binding and transmission of HIV-1 by lectin expressing T-REx cells. T-REx cells were induced to express the indicated lectins, preincubated in the presence or absence of mannan and incubated for 3 h with replication-competent NL4-3 reporter virus harboring the luciferase gene in place of nef. Thereafter, the cells were washed and either processed to analyze viral binding or transmission to target cells. Binding was assessed by lysing the cells in 1% Triton X-100 and quantification of bound antigen by p24-antigen capture ELISA. Transmission was determined by cocultivation of virus pulsed cells with CEMx174 5.25 target cells and analysis of luciferase production in the cultures 3 days after the start of the cocultivation. Results are shown as percent (%) HIV-1 binding (black bars) or transmission (white bars) by control cells, which was set as 100%. Error bars indicate SD, comparable results were obtained in an independent experiment. (B) Binding of pseudovirions harboring the HCV GP proteins to lectin expressing T-REx cells. The binding assay was carried out as described in the legend of panel A, except that HIV-1 pseudovirions harboring HCV E1 and E2 proteins in their envelope were used as input. The amount of bound virus is shown relative to the amount of input virus. A representative experiment is shown, the results were confirmed in an independent experiment. Error bars indicate SD.
Fig. 4
Fig. 4
Inhibition of lectin-mediated enhancement of ZEBOV-GP driven infection by carbohydrates and EGTA. (A) Carbohydrate inhibition of ZEBOV-GP mediated infection of lectin expressing cells. Lectin expression was induced on T-REx cells; the cells were incubated with the indicated carbohydrates (100 μg/ml final concentration) and infected with ZEBOV-GP harboring pseudotypes. A representative experiment performed in quadruplicates is shown, error bars indicate SD. The results were confirmed in two independent experiments. (B) EGTA inhibition of ZEBOV-GP mediated infection of lectin expressing cells. DC-SIGN and LSECtin expression was induced on T-REx cells, the cells treated with EGTA (5 mM final concentration) and infected with pseudovirions harboring either ZEBOV-GP or VSV-G. Input viruses were normalized for comparable luciferase production upon infection of control cells. The results are presented as fold inhibition compared to infection of target cells treated with PBS. A representative experiment is shown, error bars indicate SD. Comparable results were obtained in an independent experiment. (C) EGTA inhibition of ZEBOV-GP binding to LSECtin. Induced control cells (black line) or induced LSECtin T-REx cells (black area) were preincubated with PBS or EGTA, incubated with ZEBOV-GP-Ig fusion protein concentrated from cellular supernatants, and ZEBOV-GP-Ig binding was detected with an antibody specific for the Fc portion of the fusion protein. Reactivity is shown relative to untreated control cells incubated with secondary antibody (gray area). A representative experiment is shown, similar results were obtained in an independent experiment.
Fig. 5
Fig. 5
Glycosylation of LSECtin in transfected cells. (A) Inhibition of N-linked glycosylation by tunicamycin. DC-SIGN, LSECtin, and LSECtin variants with alterations in consensus signals for N-linked glycosylation were transiently transfected into 293T cells, the cells cultivated in the presence or absence of tunicamycin (1 μg/ml) and lectin expression was analyzed by Western blot 48 h after transfection. The results were confirmed in three independent experiments. TM, Tunicamycin. (B) Enhancement of ZEBOV-GP driven infection by LSECtin variants with defective N-linked glycosylation signals. LSECtin and the indicated LSECtin variants with defects in glycosylation signals were transiently expressed in 293T cells and lectin expression and enhancement of ZEBOV-GP driven infection analyzed in parallel. Expression was determined by staining with a LSECtin-specific antiserum and FACS analysis, while augmentation of ZEBOV-GP driven infection was assessed as described in the legend of Fig. 2A. Surface expression and augmentation of infection are shown relative to pcDNA3 transfected control cells, which was set as 100%. The average of three independent experiments performed in duplicates (surface expression) or quadruplicates (infection) is shown, error bars indicate standard error of the mean.
Fig. 6
Fig. 6
Interaction of LSECtin with the GPs of the EBOV subspecies and with replication-competent ZEBOV. (A) LSECtin enhances infection driven by the GPs of the four EBOV subspecies. T-REx cells were induced to express the indicated lectins or control vector, inoculated with infectivity normalized pseudotypes bearing the EBOV-GPs, VSV-G, or no GP (pcDNA3), and luciferase activity in cell lysates was determined as described in the legend to Fig. 2A. (B) LSECtin expression enhances infection by replication-competent ZEBOV. T-REx cell lines were seeded in chamber slides, induced to express the indicated lectins and infected with replication-competent ZEBOV. Cells producing ZEBOV antigen were detected by immunofluorescence at 24 h after infection.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alvarez C.P., Lasala F., Carrillo J., Muniz O., Corbi A.L., Delgado R. C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J. Virol. 2002;76:6841–6844. - PMC - PubMed
    1. Appelmelk B.J., van D.I., Van Vliet S.J., Vandenbroucke-Grauls C.M., Geijtenbeek T.B., Van Kooyk Y. Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells. J. Immunol. 2003;170:1635–1639. - PubMed
    1. Baribaud F., Doms R.W., Pohlmann S. The role of DC-SIGN and DC-SIGNR in HIV and Ebola virus infection: can potential therapeutics block virus transmission and dissemination? Expert Opin. Ther. Targets. 2002;6:423–431. - PubMed
    1. Bashirova A.A., Geijtenbeek T.B., van Duijnhoven G.C., Van Vliet S.J., Eilering J.B., Martin M.P., Wu L., Martin T.D., Viebig N., Knolle P.A., KewalRamani V.N., Van Kooyk Y., Carrington M. A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J. Exp. Med. 2001;193:671–678. - PMC - PubMed
    1. Becker S., Spiess M., Klenk H.D. The asialoglycoprotein receptor is a potential liver-specific receptor for Marburg virus. J. Gen. Virol. 1995;76(Pt. 2):393–399. - PubMed

Publication types