Bst2/Tetherin Is Induced in Neurons by Type I Interferon and Viral Infection but Is Dispensable for Protection against Neurotropic Viral Challenge

Abstract

In permissive mouse central nervous system (CNS) neurons, measles virus (MV) spreads in the absence of hallmark viral budding or neuronal death, with transmission occurring efficiently and exclusively via the synapse. MV infection also initiates a robust type I interferon (IFN) response, resulting in the synthesis of a large number of genes, including bone marrow stromal antigen 2 (Bst2)/tetherin/CD317. Bst2 restricts the release of some enveloped viruses, but to date, its role in viral infection of neurons has not been assessed. Consequently, we investigated how Bst2 was induced and what role it played in MV neuronal infection. The magnitude of induction of neuronal Bst2 RNA and protein following IFN exposure and viral infection was notably higher than in similarly treated mouse embryo fibroblasts (MEFs). Bst2 synthesis was both IFN and Stat1 dependent. Although Bst2 prevented MV release from nonneuronal cells, its deletion had no effect on viral pathogenesis in MV-challenged mice. Our findings underscore how cell-type-specific differences impact viral infection and pathogenesis.

Importance: Viral infections of the central nervous system can lead to debilitating disease and death. Moreover, it is becoming increasingly clear that nonrenewable cells, including most central nervous system neurons, combat neurotropic viral infections in fundamentally different ways than other rapidly dividing and renewable cell populations. Here we identify type I interferon signaling as a key inducer of a known antiviral protein (Bst2) in neurons. Unexpectedly, the gene is dispensable for clearance of neurotropic viral infection despite its well-defined contribution to limiting the spread of enveloped viruses in proliferating cells. A deeper appreciation of the importance of cell type heterogeneity in antiviral immunity will aid in the identification of unique therapeutic targets for life-threatening viral infections.

FIG 1

Bst2 RNA and protein are induced in both primary neurons and MEFs in response to interferon, but to a higher extent in neurons. Primary neurons spiked with mouse recombinant type I interferon (IFN-β) or type II interferon (IFN-γ), at a concentration of 100 U/ml, were assayed for changes in Bst2 RNA by RT-qPCR. Data are represented as fold change compared to value for untreated cells using the ΔΔCT method. n = 5 per group. Results for the unpaired t test with equal-standard-deviation samples compared to the 0-h value for the same cell type are shown. **, P < 0.005; *, P < 0.05. Results of at least 3 independent experiments are presented. Error bars represent the standard deviations among groups. (A) Bst2 RNA abundance in primary neurons. (B) Bst2 RNA abundance in primary mouse embryo fibroblasts. (C) Western blot for Bst2 and GAPDH protein from primary neurons and MEFs following IFN exposure at a dose of 100 U/ml. The image was captured using LI-COR Odyssey. (D) Coverslips of primary neurons and MEFs that were spiked with 100 U/ml of IFN for 48 h or left untreated were stained for Bst2 (green), Hoechst (blue), and β-actin (red). Each sample set is shown as Bst2 and Hoechst staining (left) or as a merged image of Bst2, Hoechst, and β-actin (right). Primary neurons without (top) or with (bottom) IFN treatment (left side) and primary MEFs without (top) or with (bottom) IFN treatment (right side) are shown.

FIG 2

Bst2 expression reduces MV egress in nonneuronal cells. Supernatants were taken from MV-infected 293T-Bst2 cells with or without tetracycline at 24, 48, and 72 h postinfection. Results of at least 3 independent experiments are presented. Error bars are used to indicate the standard deviations among groups. The number of infectious virus particles released was determined by plaque assay. Data are considered significant as determined by ANOVA. *, P < 0.05. n = 3.

FIG 3

Viral infection induces Bst2 RNA synthesis in neurons. (A) Primary neurons infected with MV (MOI = 1) or challenged with the same dose of UV-inactivated MV were assayed for Bst2 RNA levels by RT-qPCR. Data are represented as fold change over the value for untreated cells using the ΔΔCT method. n = 3 per group. Results of at least 3 independent experiments are presented. Error bars are used to indicate the standard deviations among groups. Results for unpaired t test with equal-standard-deviation samples compared to the value for the uninfected control are shown. **, P < 0.005; *, P < 0.05.

FIG 4

Bst2 induction in neurons is dependent on type I IFN signaling. (A) Primary CD46⁺ and CD46⁺/IFN-γ KO neurons were infected with MV for the indicated times and assayed for Bst2 RNA expression by RT-qPCR. (B) Primary NSE-CD46⁺, NSE-CD46⁺/Stat1 KO, and NSE-CD46⁺/IFNAR KO neurons were infected with MV for the indicated period and assayed for Bst2 RNA expression. (C) Relative levels of MV nucleoprotein RNA in indicated genotypes of primary neurons. Data are presented as fold change over the value for uninfected neurons using the ΔΔCT method. Results of at least 3 independent experiments are presented. Error bars represent the standard deviations among groups. n = 3 or 4 per group. Results for unpaired t test with equal-standard-deviation samples compared to the value for the uninfected control are shown. *, P < 0.05.

FIG 5

Bst2 expression is induced in vivo after MV infection via type I Ifn signaling. Mice of the indicated genotypes were infected intracranially with 1 × 10⁴ PFU of the MV-Edmonston. Bst2 RNA expression in whole brains was assessed 3 dpi. Data are presented as fold change over the value for uninfected mice using the ΔΔCT method. n = 3 or 4 per group. Error bars represent the standard deviations among groups. Results for npaired t test with equal-standard-deviation samples compared to the value for the uninfected control are shown. **, P < 0.005.

FIG 6

Bst2 is dispensable in vivo for survival after neuronal MV challenge. Mice of the indicated genotypes were infected intracranially with 1 × 10⁴ PFU of MV-Edmonston.