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. 2009 Feb 24;106(8):2886-91.
doi: 10.1073/pnas.0811014106. Epub 2009 Jan 28.

Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

Rachel L Kaletsky  1 , Joseph R Francica, Caroline Agrawal-Gamse, Paul Bates
Affiliations

Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

Rachel L Kaletsky et al. Proc Natl Acad Sci U S A. .

Abstract

Mammalian cells employ numerous innate cellular mechanisms to inhibit viral replication and spread. Tetherin, also known as Bst-2 or CD317, is a recently identified, IFN-induced, cellular response factor that blocks release of HIV-1 and other retroviruses from infected cells. The means by which tetherin retains retroviruses on the cell surface, as well as the mechanism used by the HIV-1 accessory protein Vpu to antagonize tetherin function and promote HIV-1 release, are unknown. Here, we document that tetherin functions as a broadly acting antiviral factor by demonstrating that both human and murine tetherin potently inhibit the release of the filovirus, Ebola, from the surface of cells. Expression of the Ebola glycoprotein (GP) antagonized the antiviral effect of human and murine tetherin and facilitated budding of Ebola particles, as did the HIV-1 Vpu protein. Conversely, Ebola GP could substitute for Vpu to promote HIV-1 virion release from tetherin-expressing cells, demonstrating a common cellular target for these divergent viral proteins. Ebola GP efficiently coimmunoprecipitated with tetherin, suggesting that the viral glycoprotein directly interferes with this host antiviral factor. These results demonstrate that tetherin is a cellular antiviral factor that restricts budding of structurally diverse enveloped viruses. Additionally, Ebola has evolved a highly effective strategy to combat this antiviral response elicited in the host during infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Tetherin inhibits budding of Ebola VLPs. (A) Human 293T cells were cotransfected with Ebola FLAG-VP40, with or without HIV vpu, and 0 to 100 ng of tetherin DNA. Cell lysates and supernatants were harvested 48 h after transfection. Virions in clarified supernatants were pelleted through sucrose. FLAG-VP40 in cell lysates and pelleted virion samples was analyzed by Western blot analysis. (B) FLAG-VP40 in cell lysates and pelleted virions was analyzed in HT1080 cells transfected with 25 ng of tetherin with or without vpu. (C) Constitutive virion release was measured from cells transfected in the presence or absence tetherin (control). Supernatants were removed, and cells were incubated with PBS (wash) or subtilisin. In all experiments, the total amount of DNA per transfection was normalized by using empty pCAGGS vector.
Fig. 2.
Fig. 2.
The Ebola glycoprotein is sufficient to overcome tetherin-restricted VLP and HIV budding. (A) 293T cells were transfected with FLAG-VP40, with or without tetherin, and empty vector (mock) or plasmid-encoded viral genes. VP40 in cell lysates and virions was analyzed as in Fig. 1. Viral gene expression in cell lysates was analyzed in B. No bands were detected in Western blot analysis of mock-transfected sample. (C) Cells were transfected with tetherin and increasing amounts of Ebola GP DNA (0 to 700 ng). Expression of GP was determined by Western blot analysis of cell lysates using GP antisera. (D) VLP budding was analyzed in HeLa cells. (E and F) HIV release was quantified by p24 ELISA of virions from 293T (E) or HeLa cells (F). HIV p24 levels in cell lysates were equivalent as determined by Western blot analysis. Error bars represent SEM of samples transfected in triplicate. (G–J) Scanning electron microscopy of HeLa cells transfected with pCAGGS alone (G) or with GFP-VP40 and pCAGGS (H), Ebola GP (I), or Vpu (J). (Scale bar: 10 μM.) (K–M) Immunofluorescence of GFP is shown in HT1080 cells transfected with GFP-VP40 alone (K) or in combination with tetherin (L and M) and Ebola GP (M). (Scale bar: 10.6 μM.) (N) VLP budding was analyzed in 293T cells transfected with VP40, murine tetherin, and the indicated plasmids.
Fig. 3.
Fig. 3.
Effect of Ebola GP mutants on tethered virions. (A and B) Cell lysates and purified virions from 293T cells transfected with FLAG-VP40, tetherin, and Ebola GP were analyzed by Western blot analysis. Expression of the various GP mutants was examined by Western blot analysis of cell lysates by using sGP antisera. (A) The effect of sGP and secGP on VLP budding was determined. (B) The GP mutants, Δmuc, ΔCl (deletion of the furin cleavage site), and GP-KKMP, were tested. (C) HIV budding in GP- and tetherin-expressing 293Ts was determined by p24 ELISA. Error bars represent SEM of samples transfected in triplicate.
Fig. 4.
Fig. 4.
Ebola GP interacts with tetherin. (A and B) 293T cells were transfected with the indicated plasmids, and RIPA lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 (A) or GP antisera (B) and Western blotted as indicated. The input represents one tenth of the immunoprecipitated sample. Tetherin appears as 3 bands representing the addition of 2 N-linked glycans. (C) Constitutively released VLPs from 293T cells were harvested (control) or treated with PBS (wash), subtilisin, or DTT. Pelleted VLPs and cell lysates were analyzed as described previously.
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