Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection - PubMed (original) (raw)

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection

Isabelle Dietrich et al. J Virol. 2011 Jun.

Abstract

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.

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Figures

Fig. 1.

Identification of a feline homologue of tetherin. (A) Alignment of the predicted amino acid sequences of feline, human, murine (rat), equine, and canine tetherins. Black bars denote transmembrane (TM) and coiled-coil regions, and an arrow marks a predicted site for glycosylphosphatidylinositol (GPI) anchor attachment. Residues are color coded as follows: light gray, hydrophobic FPMVLI; dark gray, amphiphilic WAG; green, hydrophilic neutral NQTSC; light blue, slightly basic H; blue, basic K and R; pink, slightly acidic Y; red, acidic DE. (B) Quantification of feline tetherin RNA by real-time PCR and effect of interferon treatment. IL-2-dependent CD4+ T cells, macrophages, and the FEA, AH927, CRFK, and 3201 cell lines were cultured overnight with or without feline IFN-α, IFN-γ, and IFN-ω at 103 U/ml prior to RNA extraction, cDNA preparation, and tetherin cDNA quantification. Results are expressed as mean copy numberss ± SE per cell as determined in three independent experiments (n = 3).

Fig. 2.

Effect of feline tetherin (THN) on the release of FIV and HIV. (A) FIV(VSV-G) pseudotypes bearing a GFP marker gene were prepared by transfection of 293T cells in the presence of human or feline tetherin, and viral release was quantified by titration on 293T cells. Virus production was also assessed by immunoblotting cell lysates and pelleted virions for the capsid protein p24. CON, control. (B) HIV(VSV) pseudotypes derived from a Vpu-deleted clone of HIV-1 [HIV-1 (delVpu)] were prepared in the presence of either feline or human tetherin, and HIV-1 Vpu was provided in trans. Virus release was assessed by infection of 293T cells. (C) HIV pseudotypes were also prepared from a construct with an intact Vpu (HIV-1 WT) in the presence of either feline or human tetherin, and infectivity was assessed by infection of 293T cells. (D) Ability of FIV-encoded proteins to counteract feline tetherin. FIV(VSV-G)/GFP pseudotypes were prepared in the presence or absence of both feline tetherin and a replication-defective molecular clone of FIV, CMVG8MΔpol4 (+FIVΔpol). Expression of FIV Env from CMVG8MΔpol4 was confirmed by immunoblotting with MAb VPG71.2. (E) FIV(VSV-G)/GFP pseudotypes were prepared in the presence or absence of both feline tetherin and a codon-optimized FIV OrfA, and infectivity was assayed on 293T cells. (F) HIV(VSV-G)/YFP pseudotypes were prepared in the presence or absence of both feline tetherin and human tetherin and either a codon-optimized FIV OrfA or HIV-1 Vpu, and infectivity was assayed using 293T cells.

Fig. 3.

Absence of a specific tetherin antagonist in the genome of FIV. HIV(VSV) pseudotypes derived from a Vpu-deleted clone of HIV-1 were prepared in the presence of the FIV TM219 and GL8MYA molecular clones (subtypes B and A), Env GL8, TM2, and CPG41 VR1012 expression vectors (subtypes A, B, and C), or HIV-1 (NL-43) Vpu expression vector in the presence of human tetherin (A) or feline tetherin (B). Titers of viral supernatants were determined for 293T cells and infectious titers plotted (means ± SD [n = 3]). Viral protein production in the transfected cells and virion release into the culture supernatant were monitored by immunoblotting.

Fig. 4.

Effect of stable expression of feline tetherin on FIV replication. CRFK cells were stably transduced with a retroviral vector bearing feline tetherin. (A and B) Cells were infected with the cell culture-adapted FIV-Pco COLV (A) and FIV-Fca F14 (B) strains, and virus replication was monitored by RT assay of the supernatant (means ± SE [n = 3]). (C and D) Representative fields displaying enhanced syncytium formation in tetherin-expressing cells following FIV-Fca F14 infection (D) compared with control cells (C). (E and F) Infection of CRFK cells stably expressing CD134 and either feline tetherin or synthetic feline TRIMCyp with primary isolates FIV-Fca GL8 (E) and FIV-Fca PPR (F). There was no inhibition of viral replication by tetherin in comparison with potent inhibition by TRIMCyp (mean [n = 2]).

Fig. 5.

Effect of tetherin expression on virus release and syncytium formation. (A and B) CRFK cells expressing feline tetherin were transfected with molecular clones of FIV GL8 (A) or FIV F14 (B). Virus release was monitored by RT assays (means ± SE [n = 3]). (C and D) Syncytium formation in FIV F14-transfected cells in the absence (C) or presence (D) of tetherin (representative fields observed microscopically in methylene blue/basic fuchsin-stained monolayers). (E and F) Measurement of FIV GL8 growth (E) and syncytium formation (F) in the absence or presence of tetherin. Virus release was monitored by RT assays of supernatant (means [n = 2]), while syncytium formation was observed macroscopically following staining (as described above). Representative syncytia are indicated (arrows).

Fig. 6.

Effect of interferon-ω on FIV production and syncytium formation. CRFK cells were infected with FIV-F14, and virus production was monitored by RT assays. Cells were left untreated (CON), pretreated with IFN-ω 24 h prior to infection, or treated with IFN-ω 24 h postinfection. (B) Virus protein levels were monitored in lysates of the infected cells and in culture supernatants at day 4 postinfection by immunoblotting. Induction of tetherin expression was confirmed by real-time PCR (THN CT value). (C) Representative images of syncytium formation at day 3 postinfection (phase-contrast microscopy). Dark areas packed with nuclei represent syncytia.

Fig. 7.

Distribution of FIV in infected cells. CRFK cells stably expressing feline tetherin incorporating an internal HA tag were fixed and stained for expression of FIV Env (FITC [green]) (A and D) or tetherin (Alexa Fluor 594 [red]) (B and E). As shown in merged images (C and F), nuclei were also visualized with DAPI (blue). Cells were stained either in intact form (A to C) or following detergent permeabilization (D to F). Images are representative of at least five separate fields; arrows indicate regions where Env and tetherin expression coincided.

Fig. 8.

Electron microscopy of CRFK cells infected with FIV. (A) Microvillus-rich regions in FIV-infected control cells with occasional ∼0.1-μm-diameter particles (inset, two particles [arrow] adjacent to a cross-section of a microvillus for comparison). (B and C) Aggregates of ∼0.1-μm-diameter particles aligned on the surface of tetherin-expressing cells infected with FIV. Arrows indicate ∼0.1-μm-diameter particles associated with the cell surface.

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Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection - PubMed (original) (raw)