Inhibition of Lassa and Marburg virus production by tetherin

Abstract

Recently, tetherin has been identified as an effective cellular factor that prevents the release of human immunodeficiency virus type 1. Here, we show that the production of virus-like particles induced by viral matrix proteins of Lassa virus or Marburg virus was markedly inhibited by tetherin and that N-linked glycosylation of tetherin was dispensable for this antiviral activity. Our data also suggest that viral matrix proteins or one or more components that originate from host cells are targets of tetherin but that viral surface glycoproteins are not. These results suggest that tetherin inhibits the release of a wide variety of enveloped viruses from host cells by a common mechanism.

FIG. 1.

Inhibitory effects of tetherin and its mutants against Lassa VLP release. (A) Tetherin (WT) contains an N-terminal intracellular domain (ID), a transmembrane domain (TM), a central extracellular domain (ED), and a C-terminal GPI anchor (GPI). Arrowheads indicate the predicted sites of cleavage prior to the addition of the GPI anchor. Tetherin possesses two potential N-linked glycosylation sites at positions 65 and 92 in the ED. N65A and N92A are mutants with the loss of a glycosylation site by an Asn-to-Ala substitution at positions 65 and 92, respectively. N65A/N92A is a nonglycosylated mutant with the loss of both glycosylation sites. (B and D) The Lassa virus Z and GP-C expression plasmids were cotransfected with the expression plasmid for WT or mutant tetherin or an empty vector (Control) into COS-7 cells (B) or 293T cells (D). Extracellular VLPs induced by Lassa virus Z/GP-C were pelleted from the culture fluids. Cell- or VLP-associated Z and GP-C (GP-2) were detected by Western blotting using rabbit anti-Z antiserum and mouse anti-GP-2 monoclonal antibody. WB using anti-FLAG antibody was also performed to examine the expression of WT and mutant tetherin in cells. WB for actin was done as the internal control. (C) The intensities of the bands for VLP-associated Z or GP-2 in panel B were quantified using a LAS3000 imaging system (Fujifilm). The level of Z or GP-2 in VLPs released from cells cotransfected with control vector was set to 100%. The data are shown as averages and standard deviations for three independent experiments. (E) COS-7 cells were cotransfected with the Lassa virus Z expression plasmid and the expression plasmid for tetherin (WT) or the empty vector (Control). VLPs induced by Z alone were examined by WB as described above. (F) 293T cells were cotransfected with pCLV-Z and the empty vector (left) or the expression plasmid for tetherin (right). At 48 h posttransfection, cells were observed by electron microscopy, which was performed as described previously (9). Mock, mock infected; Teth, tetherin. Bars, 500 nm.

FIG. 2.

Effects of WT and mutant tetherin expression on Marburg VLP production. (A) The Marburg virus VP40 expression plasmid was cotransfected with the expression plasmid for WT or mutant tetherin or an empty vector (Control) into COS-7 cells. Marburg virus VP40-induced VLP release from cells was analyzed by WB using rabbit anti-VP40 antiserum. WB using an anti-FLAG antibody was also performed to examine the expression of WT and mutant tetherin in cells. WB for actin was done as an internal control. (B) The intensities of the bands for VLP-associated VP40 in panel A were quantified as described in the legend to Fig. 1C. The level of VP40 in VLPs released from cells cotransfected with the control vector was set to 100%. The data are shown as averages and standard deviations for three independent experiments. Mock, mock infected; Teth, tetherin.

FIG. 3.

Enhancement of Lassa and Marburg VLP release from HeLa cells by Vpu. The expression plasmid for HIV-1 Vpu (+) or the empty vector (−) was cotransfected with the expression plasmid for Lassa virus Z (A) or Marburg virus VP40 (B) into HeLa cells. Lassa or Marburg VLP release from HeLa cells in the absence or presence of Vpu was analyzed by WB. WB using anti-Myc antibody was also performed to examine the expression of Vpu in cells. Mock, mock infected.