Broad-spectrum inhibition of retroviral and filoviral particle release by tetherin

Abstract

The expression of many putative antiviral genes is upregulated when cells encounter type I interferon (IFN), but the actual mechanisms by which many IFN-induced gene products inhibit virus replication are poorly understood. A recently identified IFN-induced antiretroviral protein, termed tetherin (previously known as BST-2 or CD317), blocks the release of nascent human immunodeficiency virus type 1 (HIV-1) particles from infected cells, and an HIV-1 accessory protein, Vpu, acts as a viral antagonist of tetherin. Here, we show that tetherin is capable of blocking not only the release of HIV-1 particles but also the release of particles assembled using the major structural proteins of a variety of prototype retroviruses, including members of the alpharetrovirus, betaretrovirus, deltaretrovirus, lentivirus, and spumaretrovirus families. Moreover, we show that the release of particles assembled using filovirus matrix proteins from Marburg virus and Ebola virus is also sensitive to inhibition by tetherin. These findings indicate that tetherin is a broadly specific inhibitor of enveloped particle release, and therefore, inhibition is unlikely to require specific interactions with viral proteins. Nonetheless, tetherin colocalized with nascent virus-like particles generated by several retroviral and filoviral structural proteins, indicating that it is present at, or recruited to, sites of particle assembly. Overall, tetherin is potentially active against many enveloped viruses and likely to be an important component of the antiviral innate immune defense.

FIG. 1.

Tetherin inhibits lentivirus VLP release. Shown is a Western blot analysis of 293T cells and the corresponding VLPs after coexpression of lentivirus Gag-Pol (A to D) or myc-tagged Gag (E) protein in the absence or presence of Vpu and various amounts of tetherin-HA. HIV-1 (A), SIV_MAC Gag (B), and SIV_AGMSab (C) proteins were revealed using an anti-HIV-1 p24CA antibody. EIAV Gag (D) was revealed with anti-EIAV serum. FIV Gag (E) was revealed with anti-myc antibodies. Numbers below each lane indicate values obtained upon densitometric scanning of VLP blots.

FIG. 2.

Tetherin inhibits alpharetrovirus, betaretrovirus, and deltaretrovirus release. Shown is a Western blot analysis of 293T cells and corresponding VLPs after the coexpression of Gag-myc or Gag-Pol proteins in the presence or absence of Vpu and various amounts of tetherin. RSV Gag (A) was revealed with anti-myc antibodies, MPMV Gag (B) and HERV-K Gag (C) were revealed using anti-CA antisera, and HTLV-1 Gag (D) was revealed with an anti-HTLV-1 p19 antibody. Numbers below each lane indicate values obtained upon densitometric scanning of VLP blots.

FIG. 3.

Tetherin inhibits spumaretrovirus VLP release. Shown is a Western blot analysis of 293T cells and the corresponding VLPs after the expression of WT PFV Gag (A), either alone or along with a PFV Env, or retargeted Lck-PFV-Gag (B) in the absence or presence of Vpu and various amounts of tetherin-HA. PFV Gag was revealed with anti-PFV human serum. Numbers below each lane indicate values obtained upon densitometric scanning of VLP blots.

FIG. 4.

Tetherin inhibits filovirus release. Shown is a Western blot analysis of 293T cells and the corresponding VLPs after the coexpression of the filovirus matrix protein Vp40 in the absence or presence of Vpu and various amounts of tetherin-HA. myc-Eb Vp40 (A) was revealed with anti-myc antibodies, while GFP-Mv Vp40 (B) was revealed with anti-GFP antibodies. Numbers below each lane indicate values obtained upon densitometric scanning of VLP blots.

FIG. 5.

Localization of nascent retrovirus and filovirus VLPs and tetherin at the plasma membrane. (A) Images showing a series of deconvolved optical sections through 293T cells stably expressing tetherin-HA and stained with an anti-HA antibody (red). Numbers in each panel indicate the z-axis distance (in μm) from the cell coverslip interface. (B) 293T cells stably expressing tetherin-HA (red) were transfected with various viral structural proteins, as indicated, fused to GFP (green) and fixed. Nuclei were stained with Hoechst stain (blue). Single deconvolved optical sections acquired at the cell-coverslip interface are shown. (C) Expanded view of a portion of the images shown in A exemplifying the localization of tetherin puncta with MLV Gag-GFP puncta and Mv Vp40 filaments. Scale bars indicate 10 μm (A) and 4 μm (B).

FIG. 6.

Quantitative analysis of colocalization between tetherin and nascent VLPs. Images were acquired at the focal plane representing the cell-coverslip interface. For each viral protein-GFP fusion, 10 pixel-square regions encompassing puncta or filaments of viral protein-GFP fluorescence were selected for three to five viral protein-GFP-expressing cells. The mean fluorescence intensities associated with viral protein-GFP (x axis) and tetherin (y axis) in each region are plotted. a.u., arbitrary units.