Identification of alternatively translated Tetherin isoforms with differing antiviral and signaling activities

Abstract

Tetherin (BST-2/CD317/HM1.24) is an IFN induced transmembrane protein that restricts release of a broad range of enveloped viruses. Important features required for Tetherin activity and regulation reside within the cytoplasmic domain. Here we demonstrate that two isoforms, derived by alternative translation initiation from highly conserved methionine residues in the cytoplasmic domain, are produced in both cultured human cell lines and primary cells. These two isoforms have distinct biological properties. The short isoform (s-Tetherin), which lacks 12 residues present in the long isoform (l-Tetherin), is significantly more resistant to HIV-1 Vpu-mediated downregulation and consequently more effectively restricts HIV-1 viral budding in the presence of Vpu. s-Tetherin Vpu resistance can be accounted for by the loss of serine-threonine and tyrosine motifs present in the long isoform. By contrast, the l-Tetherin isoform was found to be an activator of nuclear factor-kappa B (NF-κB) signaling whereas s-Tetherin does not activate NF-κB. Activation of NF-κB requires a tyrosine-based motif found within the cytoplasmic tail of the longer species and may entail formation of l-Tetherin homodimers since co-expression of s-Tetherin impairs the ability of the longer isoform to activate NF-κB. These results demonstrate a novel mechanism for control of Tetherin antiviral and signaling function and provide insight into Tetherin function both in the presence and absence of infection.

Figure 1. Alignment of Tetherin sequences and comparison to a consensus Kozak translation initiation sequence.

(A) Amino acid alignment comparing the amino-terminal cytoplasmic region of Tetherin from various mammalian species. Accession numbers- Homo sapiens: NP_004326, Pan troglodytes: NP_001177409 XP_552491, Gorilla gorilla: ADI58594, Macaca mulatta: ACV96781, Mus musculus: NP_932763 XP_134266, Rattus norvegicus: NP_937767 XP_579725, Sus scrofa: NP_001155227 (CL Sequencer; 80% limit). Highlighted conserved residues include two cytoplasmic methionine residues, M1 and M13 (arrows), and a previously characterized dual tyrosine motif (*). (B). Comparison of the nucleotide sequences at the M1 and M13 AUGs in human tetherin mRNA to a consensus Kozak translation initiation sequence. Important residues at the −3 and +1 to +4 positions are highlighted in bold black text. Human tetherin does not conform to the consensus at the −3 position. l-Tetherin and s-Tetherin refer to proteins initiated at M1 or M13 respectively. R = Purine.

Figure 2. Tetherin exists as two isoforms.

(A) Alignment of the Tetherin amino acid (Black text) and cDNA (Grey text) sequences compared to strong Kozak sequence, M1A and M13I point mutations (highlighted in black). (B) HT1080 cells transiently transfected with Tetherin encoding plasmids (WT, Strong Kozak, M1A, M13I or M1A+M13I) were lysed, treated with glycosidase (PNGase) to remove carbohydrate modifications and analyzed by Western blot using a polyclonal Tetherin antibody. l = long isoform, s = short isoform (C) Tetherin isoform expression in cell lysates from IFN stimulated and unstimulated 293T (1^st panel), unstimulated HeLa (2^nd panel) and primary CD4 T (3^rd panel) cells. Deglycosylated tetherin profiles are compared to those from transiently expressing 293T cells (4^th panel panel). l- and s- indicate the long and short Tetherin isoforms. Stable tetherin dimers (d) and an uncharacterized smaller molecular mass species (*) often observed under transient transfected conditions are indicated.

Figure 3. Tetherin isoforms have different sensitivities to viral antagonists.

(A and B) 293T cells were transfected with a constant amount of an HIV-1 Gag-Pol expression vector, a constant amount of the indicated Tetherin expression plasmid and increasing amounts of plasmids expressing the viral antagonists (A) Vpu or (B) ebolavirus GP. VLPs (Top panels) from supernatants were purified and analyzed for HIV-1 Gag p24 release. Cell lysates were probed for cellular levels of HIV Gag and Tetherin. As in Figure 2C (*) indicates an undefined species often seen upon transient Tetherin expression in 293T cells. Titration of viral antagonists was assessed using Vpu and GP specific antibodies. GAPDH is used as a lysate loading control.

Figure 4. Effect of viral antagonists on surface levels of Tetherin.

293T cells were transiently co-transfected with the indicated Tetherin plasmids in the presence viral antagonists Vpu or ebolavirus GP. Half the cells were analyzed by flow cytometry. Graph represents of the mean MFI from multiple experiments (n = 5); error bars = SEM. Surface Tetherin in the absence of viral antagonist was set to 100%. Expression of total cellular Tetherin was analyzed by Western blot of PNGase treated lysates from cells not used for flow cytometry. Reduced monomers, stable dimmers (d) and an unknown species (*) are indicated. Arrow indicates s-Tetherin isoform seen in wt+Vpu expressing cells. GAPDH used as a loading control.

Figure 5. Resistance to Vpu antagonism requires mutations in the tyrosine and serine/threonine motifs in Tetherin.

(A) Amino acid sequences of l-Tetherin mutants that disrupt the tyrosine motif (l-AxA), the serine/threonine residues (l-STS) and the combined mutant (l-SY). (B) 293T cells were transfected with HIV-1 Gag-Pol and Tetherin expression vectors plus increasing amounts of plasmids expressing the viral antagonist Vpu (25 or 100 ng). VLPs (Top panels) from supernatants were purified 48 h post transfection and analyzed for HIV-1 Gag p24 release by Western blot. Cell lysates were probed for cellular levels of HIV-1 Gag and Tetherin. As in Figure 2C (*) indicates an unknown species often seen upon transient Tetherin expression in 293T cells. Titration of Vpu was detected using anti-Vpu antibody. GAPDH is used as a lysate loading control.

Figure 6. Tetherin isoforms differentially activate NF-κB.

(A) 293T cells transiently co-transfected with either a wt, l-Tetherin, s-Tetherin or l-AxA encoding plasmid plus an NF-κB responsive firefly luciferase reporter plasmid were lysed and analyzed for luciferase activity 24 h post transfection. Myc-TRAF6 used as a positive control for NF-κB activation. NF-κB signaling experiments (n = 8 in triplicates) were analyzed by one-way ANOVA. (B) Luciferase assay for AP-1 activation was assessed as in (A) using an AP1 responsive firefly luciferase plasmid (500 ng) in the presence of the same Tetherin encoding plasmids. MEKK1 and myc-TRAF6 were used as positive controls for AP1 activation. AP1 signaling experiments (n = 4 in triplicates) were analyzed by one-way ANOVA. (C) Wt and l-Tetherin were co-transfected with increasing amounts of a FLAG epitope tagged dominant negative (DN)-IKKβ and the NF-κB responsive luciferase reporter. Myc-TRAF6 was used as a positive control (n = 3 in triplicates). Representative blot showing expression of co-transfected constructs as well as a GAPDH loading control. On the Western blots, as in Figure 2C, (*) indicates unknown species. In the graphs ns = not significant; *** p<0.001.

Figure 7. s-Tetherin modulates l-Tetherin-mediated NF-κB induction.

293T cells were transiently transfected with an NF-κB luciferase reporter and varying ratios of l- and s-Tetherin expression vectors with the total tetherin expression plasmids amount kept constant. Cells were lysed and analyzed for luciferase activity. In parallel, an l:s titration (black bars) was compared to l-Tetherin titrated with empty plasmid (white bars). wt Tetherin signaling (grey bar) was assessed during each experimental replicate (n = 9 in triplicate). Two-way ANOVA performed to assess statistical significance. A representative Western blot for Tetherin is shown for each set of titrations. *p<0.05; ***p<0.001, **** p<0.0001.