The authors have declared that no competing interests exist.
Conceived and designed the experiments: LJC PB. Performed the experiments: LJC. Analyzed the data: LJC PB. Wrote the paper: LJC PB.
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.
Regulation of innate immunity is critical to maintain a balance between control of a perceived threat and immunopathology. The interferon induced cellular factor Tetherin has been shown to restrict budding of a broad range of enveloped viruses including the human immunodeficiency virus. Though Tetherin appears to be a bona fide viral restriction factor, additional cellular functions have been observed including an involvement in actin cytoskeleton organization in polarized cells, regulating interferon secretion and signaling through nuclear factor-kappa B (NF-κB). Our studies present a mechanism by which Tetherin function is regulated at the translational level through the production of alternatively translated isoforms. The short isoform of Tetherin was observed to be significantly more resistant to HIV-1 Vpu. In contrast, the longer isoform can induce NF-κB activity, a function lacking in the short isoform. Critical NF-κB signaling residues include a dual tyrosine motif, which is only present in the long isoform. Identification of these isoforms helps to illuminate how Tetherin functions, not only as a restriction factor, but also as a signaling molecule. These data highlight a previously unappreciated level of regulation and furthers our understanding of additional Tetherin functions.
Tetherin (BST-2/CD317/HM1.24) is an interferon (IFN) induced, type II transmembrane glycoprotein which has been shown to function as an intrinsic antiviral factor that restricts release of a broad range of enveloped viruses, including members of the arenavirus
Tetherin has an unusual topology that has been shown to be critical for function as a restriction factor. Tetherin is a lipid raft resident protein anchored to the cellular membrane by a N-terminal transmembrane domain and a C-terminal GPI anchor
A number of studies have examined the ability of Tetherin to function as a cellular restriction factor; however, there is evidence that Tetherin has additional roles that may be important both in the presence and absence of viral infection. Tetherin was originally described as a marker on differentiated B cells and was suggested to be involved in B cell development
Here we identify a previously undescribed short isoform of human Tetherin generated by alternative translation initiation at an in-frame codon 33 nucleotides downstream of the canonical translation start site which results in the production of two Tetherin species. The long (l-) and short (s-) Tetherin isoforms are generated in both cultured and primary human cells. Importantly, the two isoforms have distinct biological properties. s-Tetherin, which lacks 12 residues present in the long isoform, is significantly more resistant to HIV-1 Vpu-mediated downregulation and consequently more effectively restricts HIV-1 release in the presence of Vpu than l-Tetherin. In contrast, the ebolavirus glycoprotein (GP) effectively counters both Tetherin isoforms. We also find that Tetherin is a significant activator of NF-κB. However, the Tetherin isoforms have dramatically different signaling potential, with NF-κB activation requiring a tyrosine motif found in the l-Tetherin isoform but absent in s-Tetherin. These observations provide insight into how the expression of alternatively translated Tetherin isoforms regulates multiple cellular functions and begins to shed more light on a poorly characterized signaling property.
Sequence analysis of the cytoplasmic tail of Tetherin revealed minimal amino acid conservation across species. Residues that were highly conserved included two cytoplasmic methionine residues and a dual tyrosine motif (
(A) Amino acid alignment comparing the amino-terminal cytoplasmic region of Tetherin from various mammalian species. Accession numbers-
To address whether alternatively initiated Tetherin isoforms could be produced, mutations that individually abrogated each of the AUG codons were generated in the wt cDNA (
(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 (1st panel), unstimulated HeLa (2nd panel) and primary CD4 T (3rd panel) cells. Deglycosylated tetherin profiles are compared to those from transiently expressing 293T cells (4th 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.
To address whether expression of the s-Tetherin isoform was due to translational leakage, an optimized translational initiation sequence was generated by converting the sequence at the −3 position of the upstream M1 ATG in the wt Tetherin cDNA from a pyrimidine (T) to a purine (A). Upon introduction into HT1080 cells of a vector with a strong Kozak sequence at the M1 AUG, expression of l-Tetherin was greatly enhanced while expression of s-Tetherin was almost undetectable (
To determine whether both isoforms of Tetherin are endogenously expressed, non-stimulated and interferon (IFN)-stimulated 293T, HT1080, HeLa, primary human CD4 T-cell lysates were analyzed as described above and compared to 293T cells transiently expressing the M13I and M1A mutants. As predicted from previous results, no Tetherin was seen upon analysis of deglycosylated proteins from unstimulated 293T and HT1080 cells. Interferon treatment of 293T and HT1080 cells induces Tetherin expression and upon PNGase treatment, collapses a diffuse banding pattern to two bands that migrate similarly to the two isoforms produced by transfection of l-Tetherin and s-Tetherin encoding plasmids (
Tetherin exists as a disulfide-linked dimer that is believed to represent the functional form necessary for viral restriction. Structural studies on the ectodomain suggest that Tetherin dimers may also form higher order aggregates, which may be functionally important
The cytoplasmic tail of Tetherin is dispensable for function as a viral restriction factor
(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
To determine whether the differential sensitivity to viral antagonists was a common property of the Tetherin isoforms, sensitivity to a different viral factor, the ebolavirus glycoprotein, GP, was analyzed. GP is hypothesized to rescue virus budding in Tetherin-expressing cells through a non-cytoplasmic tail-associated mechanism
To determine whether the differential particle release by the isoforms in response to Vpu was due to altered Tetherin cell surface levels, flow cytometric analysis of live cells stained for Tetherin was performed. In Vpu expressing cells, surface wt Tetherin expression followed the same trend as the total cell Tetherin protein levels (compare
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.
To further explore the basis for the relative Vpu resistance of s-Tetherin compared to l-Tetherin, a series of mutations were engineered into the 12 residue region unique to l-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
Although the tyrosine and serine/threonine mutants appear to similarly affect response to Vpu antagonism and particle release, the STS mutant Tetherin protein demonstrates resistance to Vpu-mediated degradation comparable to that of s-Tetherin (third panel,
Tetherin has been primarily characterized as a cell intrinsic viral restriction factor. However, Tetherin has been proposed to have additional activities including a role in induction of the proinflammatory response regulator 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
We next assessed whether the Tetherin isoforms had differential abilities to activate NF-κB. Unlike wt Tetherin, the truncated s-Tetherin isoform displayed no NF-κB activation (
As described above, Tetherin can assemble into hetero and homodimers of the l- and s- isoforms. Given the observations that s-Tetherin does not activate NF-κB, l-Tetherin is more potent than wt, and wt Tetherin has an intermediate signaling phenotype to that of l- and s-isoforms, we investigated whether the short isoform could modulate signaling activity of the longer species. To address this question, varying ratios of the two isoforms were assessed for their ability to activate NF-κB. As seen in
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.
Within the 12 amino acids unique to l-Tetherin are two highly conserved tyrosine residues at positions 6 and 8 (
Here we identify a previously undescribed isoform of human Tetherin generated by translation at an in-frame initiation codon 33 nucleotides downstream of the canonical translation start site, which results in the loss of a 12-amino-acid N-terminal sequence. Alternative translation initiation in eukaryotes can occur by 3 different mechanisms: internal ribosome entry, reinitiation, or leaky ribosome scanning. Our analysis indicates that the tetherin mRNA contains a “leaky” Kozak sequence around the first AUG codon (M1). Based on the scanning model of translation, this would allow the ribosome to skip over the first start codon a fraction of the time, leading to initiation at the next in frame AUG (M13). Selective disruption of each AUG and introduction of a strong Kozak at the upstream AUG confirmed that alternate sites of translation initiation accounted for each of the observed products. Conservation of an upstream leaky Kozak and a second cytoplasmic methionine in most mammalian tetherin sequences, along with our analysis of expression from rhesus and murine cDNAs, suggests that multiple isoforms exist in other species and indicate an important biologic role(s) for the two isoforms.
For many messages with alternatively translated isoforms the resultant proteins display distinct functionalities
In several other systems where alternative translation initiation produces multiple isoforms there are observed differences in the ratios of the isoforms in tissues
The importance of the shorter Tetherin isoform with increased antiviral activity or resistance to viral antagonists is strongly supported by recent evidence. While this manuscript was in preparation, a polymorphic allele that abrogated expression from the first methionine residue, and thus produced only short isoforms, was described in NZW mice. In these mice, expression of shorter Tetherin isoforms strongly correlated with decreased Friend retrovirus replication and pathogenesis
We find that the ability of s-Tetherin to retain budding HIV-1 virions and to resist Vpu more effectively than l-Tetherin appears to be due to loss of tyrosine and serine threonine motifs found in the cytoplasmic tail of the long isoform. Mutation in either motif rendered Tetherin partially resistant to Vpu. Combining these two sets of mutations recapitulated loss of Vpu sensitivity seen for s-Tetherin suggesting these represent important differences between l- and s-Tetherin. The tyrosine motif in l-Tetherin is a non-canonical trafficking signal (YXYXXV) that engages the AP-1 and AP-2 clathrin adaptors
The observation that the long isoform activates NF-κB while the short isoform shows no activity suggests that sequences within the 12 residues unique to l-Tetherin mediate signaling. Moreover, data demonstrating higher NF-κB induction by expression of the l-Tetherin isoform compared to the wt cDNA, coupled with an inhibitory affect of s-Tetherin on NF-κB activation, support a model in which homodimers of the longer isoform are responsible for signaling. Though Tetherin does not possess canonical tyrosine-based motifs involved in activating signal transduction pathways, there is evidence that dimerization of CLEC-2 allows approximation of a non-canonical tyrosine-based motif and permits signaling via Syk kinase
It has been demonstrated that Tetherin is a ligand for ILT7 on human dendritic cells and that binding initiates signaling via the ILT7–FcεRIγ complex, inhibiting production of interferon and proinflammatory cytokines by dendritic cells
293T, HT1080 and HeLa cells were maintained in high glucose DMEM+10% Cosmic Calf Serum (Hyclone). Primary CD4 T cells (obtained from the University of Pennsylvania Center for AIDS Research Immunology Core, ND307) were maintained in RPMI+10% FBS (Invitrogen). All cells were maintained at 37°C with 5% CO2.
Human tetherin cDNA in pCMV-SPORT6 was acquired from Open Biosystems. Tetherin mutants were generated using site-directed mutagenesis by PCR. Primers for site-directed mutagenesis were designed using QuikChange Primer Design Program (Agilent Technologies). The HIV-1 gag-pol encoding construct, psPAX2 was obtained from Addgene (plasmid 12260). HIV-1 vpu and ebolavirus GP cloned into pCAGGS were previously described
HT1080 cells in a 6 well were transiently transfected with Tetherin expression constructs using Lipofectamine2000 (Invitrogen) according to manufacturers instructions. For IFN induction experiments, HT1080, 293T, HeLa and primary CD4 T cells were incubated +/− 1000 U of recombinant type I IFN (PBL Interferon Source) for 48 h. Two days post-transfection/IFN treatment, cells were washed with PBS and lysed using RIPA buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 140 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% NP40). Lysates were passaged through a 25G needle before being cleared by centrifugation at 17,900× g for 15 min at 4°C. Cleared lysates were treated with either treated or not with PNGase (New England BioLabs) for 2 h, then reduced using DTT containing loading buffer. Samples were separated on a 15% Criterion gel (BioRad) and transferred to PVDF membrane. After blocking in 5% milk TBST (Tris-buffered saline+0.1% Tween), Western blots were analyzed for Tetherin expression using rabbit anti-BST2 sera (Dr. Klaus Strebel, #11721 National Institutes of Health AIDS Research and Reference Reagent Program).
2.5×105 293T cells seeded on a 24 well plate were co-transfected with the indicated tetherin construct (12.5 ng), an HIV-1 Gag-Pol expression vector pSPAX (50 ng) and increasing amounts of (25, 50 or 100 ng) of pCAGSS-vpu or pCAGGS ebolavirus GP using Lipofectamine2000. Cell lysates and supernatants were harvested 48 h post-transfection. Cell lysates were harvested in Triton X-100 buffer (50 mM Trs-HCl pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100) with Complete (Roche) protease inhibitor cocktail. Cell lysates were cleared by centrifugation at 17,900× g for 3 min at 4°C. Supernatants containing VLPs were cleared are 1700× g for 2 min at 4°C. VLPs were subsequently purified by pelleting through a 20% sucrose cushion at 40,000 in a TLA120.1 rotor (Beckman) for 30 min. VLPs were resuspended in PBS on ice for 3 h. Cleared cell lysates and resuspended VLPs were separated on 15% or 4–15% Criterion gels (BioRad) respectively before being transferred to PVDF membrane. Membranes were blocked in 5% milk in TBST for 40 min prior to incubation with primary antibody. HIV-1 Gag p24 was detected in Western blots for both VLPs and cell lysates using a monoclonal anti-p24 antibody (24-3, National Institutes of Health AIDS Research and Reference Reagent Program). The GAPDH loading control was analyzed using GAPDH antibody (Calbiochem, CB1001). HIV-1 Vpu was detected using HIV-1 pNL4-3 Vpu antiserum (969, National Institutes of Health AIDS Research and Reference Reagent Program). Ebolavirus GP was detected using rabbit antiserum against the GP1 portion of the protein
293T cells (2.5×105) seeded on a 24 well plate were transiently co-transfected with 25 ng Tetherin constructs (wt, l-Tetherin, s-Tetherin or l-AxA) and 100 ng HIV-1 vpu or ebolavirus GP. Two days post transfection, cells were washed once with 1×PBS while on the plate. Whole cells were removed using cold 1×PBS and the cells were split into equal aliquots used for flow cytometry and western blot analysis. Cells used for Western blot analysis were pelleted by centrifugation then lysed in Triton X-100 buffer. Lysates were cleared and PNGase treated prior to SDS/PAGE Western blot analysis as described above. Cells to be used for flow cytometry were resuspended in cold FACS buffer (1×PBS, 1%BSA+0.05% sodium azide) and pelleted at 2150× g. Cells were resuspended in FACS buffer+PE conjugated anti-BST2 antibody (Biolegend). After a 1 h incubation on ice, cells were washed three times with cold FACS buffer and analyzed on a FACS Calibur (BD Biosciences Immunocytometry Systems). Data analysis performed using FlowJo 9.3.1 (Tree Star).
293T cells (2.5×105) were seeded on a 24 well plate and the following day were transfected with the indicated tetherin constructs (50 ng) and pBIIX-Luciferase reporter (250 ng) using Lipofectamine2000. In experiments where the long and short isoforms were co-expressed the total amount of tetherin expression plasmid was kept constant at 50 ng. At 30 h post-transfection, cell lysates were harvested in Triton X-100 lysis buffer. Lysates were transferred to a black flat bottom 96-well plate. Luciferase Assay System substrate (Promega E1501) was added to the lysates according to manufacturers directions. Samples were analyzed in a Luminoskan Ascent microplate luminometer (Thermo Scientific). Statistical analysis was performed using PRISM (GraphPad).
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We thank Rachel Kaletsky for generating the AU1 s-Tetherin epitope tagged construct, and Sunny Shin and Michael May for supplying plasmids.