Tetherin is a key effector of the antiretroviral activity of type I interferon in vitro and in vivo

Abstract

Tetherin (Bst-2 CD317) is a cell-surface protein whose expression is induced by IFNα. Although tetherin expression causes the retention of retrovirus particles on the surface of infected cells, it is not known whether tetherin inhibits retroviral replication or pathogenesis in vivo. Mutation of tetherin antagonists often has little effect on retroviral replication in vitro, and, although tetherin can reduce the yield of extracellular viral particles, some studies suggest that tetherin actually enhances direct cell-to-cell viral transmission. We generated tetherin-deficient mice to determine the effect of this protein on murine retrovirus replication and pathogenesis. We find that tetherin markedly inhibits the replication of Moloney murine leukemia virus (Mo-MLV) and is required for the antiretroviral activity of IFNα to be fully manifested in vitro. Surprisingly, Mo-MLV replication and disease progression was not significantly different in WT and tetherin-deficient mice, but this finding was explained by the fact that Mo-MLV infection did not induce detectable tetherin expression on candidate target cells in vivo. Indeed, IFNα induction was required to reveal the anti-Mo-MLV activity of tetherin in vivo. Moreover, LP-BM5, an MLV strain that has been demonstrated to induce immune activation and IFNα expression, achieved higher levels of viremia and induced exaggerated pathology in tetherin-deficient mice. These data indicate that tetherin is a bona fide antiviral protein and can reduce retroviral replication and disease in vivo.

Fig. 1.

Tetherin potently inhibits Mo-MLV replication in vitro. (A) Accumulation of Mo-MLV, in focus-forming units/milliliter (FFU/mL), in the supernatant of NIH/3T3 cells infected at a multiplicity of infection of 0.005 in the presence or absence of 100 U/mL IFNα. (B) Flow cytometric analysis of tetherin expression on untreated (shaded histogram) or 100 U/mL IFNα-treated (unshaded histogram) NIH/3T3 cells. (C) Tetherin levels on a panel of six NIH/3T3 cell clones stably expressing tetherin (colored histograms) or empty vector (shaded histogram). (D) Mo-MLV replication in cell lines from C and on IFNα-treated cells (Vector + IFN). (E) Mo-MLV accumulation at day 4 plotted against tetherin expression [mean fluorescence intensity (MFI)] for the cell lines in C and D. Note that the colors, symbols, and line styles in C–E are matched.

Fig. 2.

Tetherin is required for the full antiretroviral activity of IFNα in vitro. (A) Tetherin expression on WT (+/+) and tetherin-deficient (−/−) MEFs that were untreated (shaded histogram) or treated with 100 U/mL IFNα for 24 h (unshaded histogram). (B and C) Mo-MLV replication in WT (B) or tetherin-deficient (C) MEFs, in the presence (+IFNα) and absence (No IFN) of 100 U/mL IFNα assayed as in Fig. 1.

Fig. 3.

No effect of tetherin deficiency on Mo-MLV infection and pathogenesis in vivo. (A) Shown are numbers of infected cells, determined by using infectious center assays, performed with cells harvested from Mo-MLV–infected mouse tissues at 8 d after infection. (B) Kaplan–Meier survival curves of Mo-MLV–infected mice: +/+, n = 12; +/−, n = 10; −/−, n = 9. (C) Flow cytometric analysis of tetherin expression on bone marrow-derived cells from mock-infected (shaded histograms) or Mo-MLV–infected (unshaded histograms) mice at 12 d after infection. (D) Kaplan–Meier survival curves of Mo-MLV–infected WT (n = 7) or IFNAR1-deficient (n = 7) mice.

Fig. 4.

Endogenous tetherin is induced by type I IFN in vivo. (A) One-week-old mice were mock-treated (shaded histograms), IFNα-treated (solid line), or poly(I:C)-treated (dashed line), and bone marrow was harvested at 24 h later for flow cytometric analysis of tetherin levels on various cell types. (B) Quantitation of tetherin expression as in A from three donors. Error bars indicate 1 SD. (C–G) WT (+/+), tetherin-deficient (−/−), and IFNAR1-deficient (IFNAR1−/−) mice were treated with poly(I:C), and FACS analysis was performed on splenocytes 24 h later. Shaded histograms represent control PBS-treated animals, and unshaded histograms represent poly(I:C)-treated animals. Tetherin expression was assessed on total splenocytes (C), and expression of the indicated activation markers was assessed on CD3⁺ (D), B220⁺ (E), and CD11c⁺ (F) cells.

Fig. 5.

Tetherin induction reduces Mo-MLV plasma viremia in vivo. Mo-MLV–infected mice were left untreated and plasma viremia was measured at 12 d after infection (A) or were treated with poly(I:C) at 12 d after infection and plasma viremia was assayed 24 h later (B). Horizontal lines indicate the mean of each data set.

Fig. 6.

Tetherin restricts LP-BM5 MLV replication and pathogenesis in vivo. (A) LP-BM5 replication in NIH/3T3 cells stably expressing tetherin or vector alone, assayed as in Fig. 1A. (B) Plasma viremia, measured by focal immunoassay, in LP-BM5–infected mice over the course of 12 wk of infection. (C–F) Weights of lymphoid tissues from LP-BM5–infected animals (white bars; +/+, n = 8; −/−, n = 8) harvested at 14 wk after infection or age-matched, uninfected animals (black bars; +/+, n = 3; −/−, n = 2). Error bars indicate 1 SE.